[go: up one dir, main page]
More Web Proxy on the site http://driver.im/

EP4050027A2 - Polypeptides se liant au cx3cr1 - Google Patents

Polypeptides se liant au cx3cr1 Download PDF

Info

Publication number
EP4050027A2
EP4050027A2 EP22168124.0A EP22168124A EP4050027A2 EP 4050027 A2 EP4050027 A2 EP 4050027A2 EP 22168124 A EP22168124 A EP 22168124A EP 4050027 A2 EP4050027 A2 EP 4050027A2
Authority
EP
European Patent Office
Prior art keywords
seq
amino acid
polypeptide
cx3cr1
acid sequence
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22168124.0A
Other languages
German (de)
English (en)
Other versions
EP4050027A3 (fr
Inventor
Alisa K. WATERMAN
Erik Depla
Toon Laeremans
Diane Van Hoorick
Cedric Jozef Néotére VERVERKEN
Sanjaya Singh
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Ablynx NV
Original Assignee
Ablynx NV
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Family has litigation
First worldwide family litigation filed litigation Critical https://patents.darts-ip.com/?family=47884535&utm_source=google_patent&utm_medium=platform_link&utm_campaign=public_patent_search&patent=EP4050027(A2) "Global patent litigation dataset” by Darts-ip is licensed under a Creative Commons Attribution 4.0 International License.
Application filed by Ablynx NV filed Critical Ablynx NV
Publication of EP4050027A2 publication Critical patent/EP4050027A2/fr
Publication of EP4050027A3 publication Critical patent/EP4050027A3/fr
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2866—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against receptors for cytokines, lymphokines, interferons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/02—Drugs for disorders of the nervous system for peripheral neuropathies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
    • C07H21/04—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids with deoxyribosyl as saccharide radical
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/715—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons
    • C07K14/7158—Receptors; Cell surface antigens; Cell surface determinants for cytokines; for lymphokines; for interferons for chemokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A61K39/3955—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals against proteinaceous materials, e.g. enzymes, hormones, lymphokines
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/22—Immunoglobulins specific features characterized by taxonomic origin from camelids, e.g. camel, llama or dromedary
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/24—Immunoglobulins specific features characterized by taxonomic origin containing regions, domains or residues from different species, e.g. chimeric, humanized or veneered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/35—Valency
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/52—Constant or Fc region; Isotype
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/565—Complementarity determining region [CDR]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/50—Immunoglobulins specific features characterized by immunoglobulin fragments
    • C07K2317/56—Immunoglobulins specific features characterized by immunoglobulin fragments variable (Fv) region, i.e. VH and/or VL
    • C07K2317/569—Single domain, e.g. dAb, sdAb, VHH, VNAR or nanobody®
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/70—Immunoglobulins specific features characterized by effect upon binding to a cell or to an antigen
    • C07K2317/76—Antagonist effect on antigen, e.g. neutralization or inhibition of binding
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/92—Affinity (KD), association rate (Ka), dissociation rate (Kd) or EC50 value
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/90—Immunoglobulins specific features characterized by (pharmaco)kinetic aspects or by stability of the immunoglobulin
    • C07K2317/94—Stability, e.g. half-life, pH, temperature or enzyme-resistance
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to CX3CR1-binding polypeptides, in particular polypeptides comprising specific immunoglobulin domains.
  • the invention also relates to nucleic acids encoding such polypeptides; to methods for preparing such polypeptides; to host cells expressing or capable of expressing such polypeptides; to compositions comprising such polypeptides; and to uses of such polypeptides or such compositions, in particular for prophylactic, therapeutic and diagnostic purposes.
  • CX3CR1 is a G-protein coupled integral membrane protein, which is a chemokine receptor. It is predominantly expressed on cell types such as monocytes, dendritic cells and T cells that have been associated with the initiation and progression of atherosclerotic plaques. It is upregulated on monocytes by oxidized lipids and mediates migration of these cells into and survival within plaques. Its unique ligand fractalkine (FKN) is expressed on the surface of vascular endothelial and smooth muscle cells in lesions where it modulates leukocyte adhesion. Fractalkine is also released into the circulation by proteolytic cleavage where it functions as a chemotactic agent.
  • FKN unique ligand fractalkine
  • CX3CR1 variant (V249I/T280M) with decreased activity has been shown to be associated with a lower risk of cardiovascular disease (coronary heart disease, cerebrovascular disease or peripheral vascular disease)( McDermott, 2001; Circ Res 89:401 ), coronary artery disease (angiographic evidence of stenosis) ( McDermott, 2003; J. Clin. Invest. 111:1241 ), and carotid artery occlusive disease ( Ghilardi, 2004; Stroke 35:1276 ).
  • CX3CR1 co-localized with fractalkine which showed enhanced immunostaining by polyclonal antibodies within atherosclerotic plaques ( Wong, 2002 Cardiovasc. Path. 11:332 ). No fractalkine staining was observed in non-plaque arterial regions.
  • CX3CR1 is involved in cardiovascular diseases and the modulation of its activity could provide promising therapies.
  • antagonist molecules against CX3CR1 with beneficial pharmacological properties which can be used as therapeutic agents to treat diseases, in particular cardiovascular diseases in humans.
  • one aim of the present invention is to provide anti-CX3CR1 antagonist molecules, in particular anti-CX3CR1 antagonist molecules, which have high binding affinity to CX3CR1.
  • a further aim of the present invention is to provide anti-CX3CR1 antagonist molecules, which have high specificity for CX3CR1.
  • a further aim of the present invention is to provide anti-CX3CR1 antagonists, which have potent activity.
  • a further aim of the present invention is to provide anti-CX3CR1 antagonists, which have a favorable bioavailability and half-life.
  • a further aim of the present invention is to provide anti-CX3CR1 antagonists, which have favorable biophysical properties.
  • aims of the present invention include combinations of any of the aims set forth above.
  • the invention provides polypeptides which bind to human CX3CR1 and are capable of blocking the binding of human fractalkine to human CX3CR1.
  • the polypeptide is an immunoglobulin comprising an antigen-binding domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1.
  • the polypeptide comprises one or more anti-CX3CR1 immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1.
  • polypeptide of the present invention is characterized by one or more of the following properties:
  • a polypeptide of the present invention comprises an anti-CX3CR1 immunoglobulin single variable domain and further comprises a half-life extending moiety, for example an albumin binding moiety, a polyethylene glycol molecule or a Fc domain.
  • a polypeptide of the present invention comprises two or more anti-CX3CR1 immunoglobulin single variable domains.
  • the two anti-CX3CR1 immunoglobulin single variable domains are covalently linked by a linker peptide.
  • the two anti-CX3CR1 immunoglobulin single variable domains in a polypeptide of the present invention have the same amino acid sequence.
  • the two anti-CX3CR1 immunoglobulin single variable domains in a polypeptide of the present invention have different amino acid sequences.
  • a polypeptide of the present invention comprises two anti-CX3CR1 immunoglobulin single variable domains and further comprises a half-life extending moiety, for example an albumin binding moiety, a polyethylene glycol molecule or a Fc domain.
  • a polypeptide of the present invention comprises a first anti-CX3CR1 immunoglobulin single variable domain covalently linked to an albumin binding moiety by a first linker peptide, wherein said albumin binding moiety is further covalently linked to a second anti-CX3CR1 immunoglobulin single variable domain by a second linker peptide.
  • a polypeptide of the present invention comprises an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide.
  • such polypeptide comprising an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide is provided as a dimer, for example through disulfide bridges.
  • polypeptides of the present invention are used for the prevention, treatment, alleviation and/or diagnosis of CX3CR1-associated diseases, disorders or conditions, in particular cardiovascular diseases, such as atherosclerosis.
  • the present invention provides:
  • the antigen-binding domain of a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a conventional 4-chain antibody such as an IgG, IgM, IgA, IgD or IgE molecule; known in the art
  • a Fab fragment, a F(ab')2 fragment, an Fv fragment such as a disulphide linked Fv or a scFv fragment, or a diabody (all known in the art) derived from such conventional 4-chain antibody would normally not be regarded as an immunoglobulin single variable domain, as, in these cases, binding to the respective epitope of an antigen would normally not occur by one (single) immunoglobulin domain but by a pair of (associating) immunoglobulin domains such as light and heavy chain variable domains, i.e. by a VH-VL pair of immunoglobulin domains, which jointly bind to an epitope of the
  • VHH domains also known as VHHs, V H H domains, VHH antibody fragments, and VHH antibodies
  • VHH domains have originally been described as the antigen binding immunoglobulin (variable) domain of "heavy chain antibodies” (i.e. of "antibodies devoid of light chains”; Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: “Naturally occurring antibodies devoid of light chains”; Nature 363, 446-448 (1993 )).
  • VHH domain has been chosen in order to distinguish these variable domains from the heavy chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as “V H domains” or “VH domains”) and from the light chain variable domains that are present in conventional 4-chain antibodies (which are referred to herein as "V L domains” or “VL domains”).
  • VHH domains can specifically bind to an epitope without an additional antigen binding domain (as opposed to VH or VL domains in a conventional 4-chain antibody, in which case the epitope is recognized by a VL domain together with a VH domain).
  • VHH domains are small, robust and efficient antigen recognition units formed by a single immunoglobulin domain.
  • VHH domain VHH, V H H domain, VHH antibody fragment, VHH antibody, as well as “Nanobody ® " and "Nanobody ® domain”
  • “Nanobody” being a trademark of the company Ablynx N.V.; Ghent; Belgium
  • N.V. being a trademark of the company Ablynx N.V.; Ghent; Belgium
  • “Nanobody” being a trademark of the company Ablynx N.V.; Ghent; Belgium
  • immunoglobulin single variable domains having the structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and specifically binding to an epitope without requiring the presence of a second immunoglobulin variable domain
  • hallmark residues as defined in e.g. WO2009/109635 , Fig. 1.
  • VHH domains are numbered according to the general numbering for V H domains given by Kabat et al. ("Sequence of proteins of immunological interest", US Public Health Services, NIH Bethesda, MD, Publication No. 91 ), as applied to VHH domains from Camelids, as shown e.g. in Figure 2 of Riechmann and Muyldermans, J. Immunol. Methods 231, 25-38 (1999 ). According to this numbering,
  • the total number of amino acid residues in each of the CDRs may vary and may not correspond to the total number of amino acid residues indicated by the Kabat numbering (that is, one or more positions according to the Kabat numbering may not be occupied in the actual sequence, or the actual sequence may contain more amino acid residues than the number allowed for by the Kabat numbering).
  • the numbering according to Kabat may or may not correspond to the actual numbering of the amino acid residues in the actual sequence.
  • the total number of amino acid residues in a VHH domain will usually be in the range of from 110 to 120, often between 112 and 115. It should however be noted that smaller and longer sequences may also be suitable for the purposes described herein.
  • VHH domains (which have been "designed" by nature to functionally bind to an antigen without the presence of, and without any interaction with, a light chain variable domain) can function as a single, relatively small, functional antigen-binding structural unit, domain or polypeptide.
  • VHH domains from the VH and VL domains of conventional 4-chain antibodies, which by themselves are generally not suited for practical application as single antigen-binding proteins or immunoglobulin single variable domains, but need to be combined in some form or another to provide a functional antigen-binding unit (as in for example conventional antibody fragments such as Fab fragments; in scFv's, which consist of a VH domain covalently linked to a VL domain).
  • VHH domains - either alone or as part of a larger polypeptide - offers a number of significant advantages over the use of conventional VH and VL domains, scFv's or conventional antibody fragments (such as Fab- or F(ab')2-fragments):
  • VHH domains derived from camelids can be "humanized" by replacing one or more amino acid residues in the amino acid sequence of the original VHH sequence by one or more of the amino acid residues that occur at the corresponding position(s) in a VH domain from a conventional 4-chain antibody from a human being.
  • a humanized VHH domain can contain one or more fully human framework region sequences, and, in an even more specific embodiment, can contain human framework region sequences derived from DP-29, DP-47, DP-51, or parts thereof, optionally combined with JH sequences, such as JH5. f2)
  • Domain antibodies also known as “Dab”s, “Domain Antibodies”, and “dAbs” (the terms “Domain Antibodies” and “dAbs” being used as trademarks by the GlaxoSmithKline group of companies) have been described in e.g.
  • Domain antibodies essentially correspond to the VH or VL domains of noncamelid mammalians, in particular human 4-chain antibodies.
  • specific selection for such antigen binding properties is required, e.g. by using libraries of human single VH or VL domain sequences.
  • Domain antibodies have, like VHHs, a molecular weight of approximately 13 to approximately 16 kDa and, if derived from fully human sequences, do not require humanization for e.g. therapeutical use in humans. As in the case of VHH domains, they are well expressed also in prokaryotic expression systems, providing a significant reduction in overall manufacturing cost.
  • Domain antibodies as well as VHH domains, can be subjected to affinity maturation by introducing one or more alterations in the amino acid sequence of one or more CDRs, which alterations result in an improved affinity of the resulting immunoglobulin single variable domain for its respective antigen, as compared to the respective parent molecule.
  • Affinity-matured immunoglobulin single variable domain molecules of the invention may be prepared by methods known in the art, for example, as described by Marks et al., 1992, Biotechnology 10:779-783 , or Barbas, et al., 1994, Proc. Nat. Acad.
  • epitopes refer to the part of a macromolecule, such as a polypeptide, that is recognized by antigen-binding molecules, such as conventional antibodies or the polypeptides of the invention, and more particularly by the antigen-binding site of said molecules. Epitopes define the minimum binding site for an immunoglobulin, and thus represent the target of specificity of an immunoglobulin.
  • an antigen-binding molecule such as a conventional antibody or a polypeptide of the invention
  • the part of an antigen-binding molecule (such as a conventional antibody or a polypeptide of the invention) that recognizes the epitope is called a paratope.
  • the term "biparatopic" (antigen-)binding molecule or "biparatopic” polypeptide as used herein shall mean a polypeptide comprising a first immunoglobulin single variable domain and a second immunoglobulin single variable domain as herein defined, wherein these two variable domains are capable of binding to two different epitopes of one antigen, which epitopes are not normally bound at the same time by one monospecific immunoglobulin, such as e.g. a conventional antibody or one immunoglobulin single variable domain.
  • Biparatopic polypeptides can be composed of variable domains which have different epitope specificities, and do not contain mutually complementary variable domain pairs which bind to the same epitope. The two variable domains do therefore not compete with each other for binding to the target.
  • a polypeptide such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, a polypeptide of the invention, or generally an antigen binding molecule or a fragment thereof
  • a polypeptide that can "bind to” or “specifically bind to”, that "has affinity for” and/or that "has specificity for” a certain epitope, antigen or protein (or for at least one part, fragment or epitope thereof) is said to be “against or “directed against “ said epitope, antigen or protein or is a "binding" molecule with respect to such epitope, antigen or protein, or is said to be “anti”- epitope, "anti”-antigen or “anti”-protein (e.g anti-CX3CR1).
  • the term "specificity" refers to the number of different types of antigens or epitopes to which a particular antigen-binding molecule or antigen-binding protein (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide of the invention) can bind.
  • the specificity of an antigen-binding protein can be determined based on its affinity and/or avidity.
  • the affinity represented by the equilibrium constant for the dissociation of an antigen with an antigen-binding protein (KD) is a measure for the binding strength between an epitope and an antigen-binding site on the antigen-binding protein: the lesser the value of the KD, the stronger the binding strength between an epitope and the antigen-binding molecule (alternatively, the affinity can also be expressed as the affinity constant (KA), which is 1/KD).
  • affinity can be determined in a manner known per se, depending on the specific antigen of interest.
  • Avidity is the measure of the strength of binding between an antigen-binding molecule (such as an immunoglobulin, an antibody, an immunoglobulin single variable domain, or a polypeptide of the invention) and the pertinent antigen. Avidity is related to both the affinity between an epitope and its antigen binding site on the antigen-binding molecule and the number of pertinent binding sites present on the antigen-binding molecule. l) Amino acid residues will be indicated according to the standard three-letter or one-letter amino acid code, as generally known and agreed upon in the art.
  • amino acid difference refers to insertions, deletions or substitutions of the indicated number of amino acid residues at a position of the reference sequence, compared to a second sequence.
  • substitution(s) will preferably be conservative amino acid substitution(s), which means that an amino acid residue is replaced with another amino acid residue of similar chemical structure and which has little or essentially no influence on the function, activity or other biological properties of the polypeptide.
  • conservative amino acid substitutions are well known in the art, for example from WO 98/49185 , wherein conservative amino acid substitutions preferably are substitutions in which one amino acid within the following groups (i) - (v) is substituted by another amino acid residue within the same group: (i) small aliphatic, nonpolar or slightly polar residues: Ala, Ser, Thr, Pro and Gly; (ii) polar, negatively charged residues and their (uncharged) amides: Asp, Asn, Glu and GIn; (iii) polar, positively charged residues: His, Arg and Lys; (iv) large aliphatic, nonpolar residues: Met, Leu, Ile, Val and Cys; and (v) aromatic residues: Phe, Tyr and Trp.
  • Particularly preferred conservative amino acid substitutions are as follows:
  • polypeptides of the invention have specificity for human CX3CR1.
  • the polypeptides of the invention preferably bind to human CX3CR1 (SEQ ID NO: 255).
  • polypeptides of the present invention also bind to cynomolgus CX3CR1 (SEQ ID NO: 256).
  • the invention provides novel pharmaceutically active agents for the prevention, treatment, alleviation and/or diagnosis of CX3CR1 associated diseases, disorders or conditions, such as cardiovascular diseases.
  • the invention provides polypeptides which bind to human CX3CR1 and are capable of blocking the binding of human fractalkine to human CX3CR1.
  • the polypeptide is an immunoglobulin comprising an antigen-binding domain comprising three complementarity determining regions CDR1, CDR2 and CDR3, wherein said immunoglobulin binds to human CX3CR1 and is capable of blocking the binding of human fractalkine to human CX3CR1.
  • the polypeptide comprises one or more anti-CX3CR1 immunoglobulin single variable domain, wherein said polypeptide is capable of blocking the binding of human fractalkine to human CX3CR1.
  • polypeptide of the present invention is characterized by one or more of the following properties:
  • a polypeptide of the present invention further comprises a half-life extending moiety, for example an albumin binding moiety, a polyethylene glycol molecule or a Fc domain.
  • a polypeptide of the present invention comprises two or more anti-CX3CR1 immunoglobulin single variable domains.
  • the two anti-CX3CR1 immunoglobulin single variable domains are covalently linked by a linker peptide.
  • the two anti-CX3CR1 immunoglobulin single variable domains in a polypeptide of the present invention have the same amino acid sequence.
  • the two anti-CX3CR1 immunoglobulin single variable domains in a polypeptide of the present invention have different amino acid sequences.
  • a polypeptide of the present invention comprises two anti-CX3CR1 immunoglobulin single variable domains and further comprises a half-life extending moiety, for example an albumin binding moiety, a polyethylene glycol molecule or a Fc domain.
  • a polypeptide of the present invention comprises a first anti-CX3CR1 immunoglobulin single variable domain covalently linked to an albumin binding moiety by a first linker peptide, wherein said albumin binding moiety is further covalently linked to a second anti-CX3CR1 immunoglobulin single variable domain by a second linker peptide.
  • a polypeptide of the present invention comprises an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide.
  • such polypeptide comprising an anti-CX3CR1 immunoglobulin single variable domain covalently linked to a Fc domain by a linker peptide is provided as a dimer, for example through disulfide bridges.
  • Single variable domains of the present invention were identified from a library expressing single variable domains (VHH) derived from a llama immunized with DNA encoding human CX3CR1.
  • VHH single variable domains
  • the phage library was panned on hCX3CR1 viral lipoparticles and binding phage were screened for their ability to compete for receptor binding with Alexa-fluor labeled fractalkine (AF-FKN).
  • AF-FKN Alexa-fluor labeled fractalkine
  • an immunoglobulin single variable domain of the present invention consists essentially of four framework regions (FR1, FR2, FR3 and FR4) and three complementary determining regions (CDR1, CDR2 and CDR3).
  • the immunoglobulin single variable domain has the structure FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.
  • the immunoglobulin single variable domain is an antibody domain.
  • the CDR3 of a polypeptide of the present invention in particular a immunoglobulin single domain of the present invention has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 as set forth in SEQ ID NO: 197, wherein:
  • the CDR3 of a polypeptide of the present invention in particular a immunoglobulin single domain of the present invention, has the amino acid sequence of Asp-Xaa1-Arg-Arg-Gly-Trp-Xaa2-Xaa3-Xaa4-Xaa5 as set forth in SEQ ID NO: 197, wherein:
  • the CDR3 of a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the amino acid sequence of Asp-Pro-Arg-Arg-Gly-Trp-Asp-Thr-Arg-Tyr as set forth in SEQ ID NO: 186.
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the following CDR1, CDR2 and CDR3:
  • a polypeptide of the present invention in particular a immunoglobuling single domain of the present invention, has the following CDR1, CDR2 and CDR3, wherein:
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the following CDR1, CDR2 and CDR3:
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the following CDR1, CDR2 and CDR3:
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the following CDR1, CDR2 and CDR3:
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the following CDR1, CDR2 and CDR3:
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the CDR1, CDR2 and CDR3 set forth in:
  • a polypeptide of the present invention in particular an immunoglobuling single domain of the present invention, has the CDR1, CDR2 and CDR3 set forth in:
  • Table 1 Representative polypeptides of the present invention having the CDRs described above are shown in Tables 1, 2, 3 (representative polypeptides of families 101, 9 and 13, respectively) and 4 (representative polypeptides of optimized variants of family 101.
  • Table 1 Family 101 Nanobody SEQ CDR1* SEQ CDR1 CDR2* SEQ CDR2 CDR3* SEQ CDR3 1 141 162 186 2 141 163 187 3 141 164 186 4 141 164 186 5 141 164 186 6 142 165 188 7 141 164 186 8 142 165 188 9 141 166 186 10 141 164 186 11 141 167 189 12 141 164 186 13 141 166 186 14 141 167 186 15 142 165 188 16 141 163 187 17 142 165 188 18 141 167 186 19 141 167 186 20 141 164 186 21 141 166 186 22 141 167 186 23
  • sequence numbers in the table refer to the sequences in the sequence listing of the instant application.
  • Table 2 Family 9 Nanobody SEQ CDR1* SEQ CDR1 CDR2* SEQ CDR2 CDR3* SEQ CDR3 49 146 176 191 50 146 176 191 51 146 177 191 52 146 177 191 *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (Eds.), Springer Verlag Heidelberg Berlin, 2010.
  • the sequence numbers in the table (SEQ) refer to the sequences in the sequence listing of the instant application.
  • SEQ The sequence numbers in the table (SEQ) refer to the sequences in the sequence listing of the instant application.
  • Table 4 Optimized variants Nanobody SEQ CDR1 SEQ CDR1 CDR2 SEQ CDR2 CDR3 SEQ CDR3 1 141 162 186 121 141 162 186 122 141 162 186 123 141 162 186 124 141 162 186 125 141 162 186 126 141 162 186 127 141 162 186 128 141 162 186 129 141 162 186 130 141 162 186 131 141 214 186 132 141 215 186 133 141 216 186 134 141 217 186 135 141 218 186 136 141 219 186 137 141 220 186 138 213 214 186 139 213 221 186 140 141 162 186 *CDR sequences were determined according to Antibody Engineering, vol 2 by Konetermann & Dübel (
  • the present invention provides polypeptides having one or more VHH domains.
  • a VHH domain of the present invention comprises or essentially consists of the sequence set forth in:
  • VHH domain of the present invention comprises or essentially consists of the sequence set forth in:
  • VHH domain of the present invention comprises or essentially consists of the sequence set forth in:
  • a VHH domain of the present invention comprises or essentially consists of the amino acid sequence set forth in any one of SEQ ID NO: 121-140, or SEQ ID NO: 222-224.
  • a VHH domain of the present invention comprises or essentially consists of the amino acid sequence set forth in any one of SEQ ID NO: 138-140.
  • a VHH domain of the present invention comprises or essentially consists of the amino acid sequence set forth in any one of SEQ ID NO: 222-224.
  • VHH domains of the present invention are shown in Table 5 and representative optimized VHH domains of the present invention are shown in Table 6 below: Table 5: VHH domains SEQ ID NO: 1-48 are VHH domains of family 101. SEQ ID NO: 49-52 are VHH domains of family 9. SEQ ID NO: 53-120 are VHH domains of family 13.
  • CX3CR1BII PMP66B02 SEQ ID NO: 1 CX3CR1BII PMP54A12 SEQ ID NO: 2 CX3CR1BII PMP54A3 SEQ ID NO: 3 CX3CR1BII PMP54A4 SEQ ID NO: 4 CX3CR1BII PMP54A5 SEQ ID NO: 5 CX3CR1BII PMP54A7 SEQ ID NO: 6 CX3CR1BII PMP54B1 SEQ ID NO: 7 CX3CR1BII PMP54B2 SEQ ID NO: 8 CX3CR1BII PMP54B3 SEQ ID NO: 9 CX3CR1BII PMP54B5 SEQ ID NO: 10 CX3CR1BII PMP54D5 SEQ ID NO: 11 CX3CR1BII PMP54D8 SEQ ID NO: 12 CX3CR1BII PMP54F6 SEQ ID NO: 13 CX3CR1BII PMP54G3 SEQ ID NO: 14 CX
  • a polypeptide according to the present invention in particular an immunoglobulin single variable domain of the present invention, is humanized and/or optimized for stability, potency, manufacturability and/or similarity to human framework regions.
  • the polypeptide is humanized and/or sequence optimized in one or more of the following positions (according to Kabat numbering): 1, 11, 14, 16, 74, 83, 108.
  • the polypeptide comprises one or more of the following mutations: E1D, S11L, A14P, E16G, A74S, K83R, Q108L.
  • one or more framework regions of a polypeptide according to the present invention are humanized and/or sequence optimized.
  • a polypeptide according to the present invention in particular an immunoglobulin single variable domain of the present invention, comprises framework regions (FR) for example as set forth below:
  • Human immunoglobulin framework region sequences that can also be used as framework region sequences for the immunoglobulin single variable domains as described above are known in the art. Also known in the art are methods for humanizing framework regions of immunoglobulin single variable domains derived from species other than humans.
  • one or more CDR regions of a polypeptide according to the present invention, in particular an immunoglobulin single variable domain of the present invention is humanized and/or sequence optimized.
  • a polypeptide according to the present invention, in particular an immunoglobulin single variable domain of the present invention is humanized and/or sequence optimized in one or more of the following positions (according to Kabat numbering): 52, 53.
  • a polypeptide according to the present invention in particular an immunoglobulin single variable domain of the present invention, comprises one or more of the following mutations: N52S, S53T.
  • a polypeptide according to the present invention in particular an immunoglobulin single variable domain of the present invention, comprises a CDR2 selected from any one of SEQ ID NO's: 214-221.
  • Table 7a shows the FR1-CDR1-FR2-CRD2 of the sequence optimized variants
  • table 7b shows FR3-CDR3-FR4-CDR4 of said variants.
  • SEQ sequence numbers in the tables
  • a polypeptide of the invention can additionally contain modifications such as glycosyl residues, modified amino acid side chains, and the like.
  • polypeptides of the invention are preferably directed against human CX3CR1, whereas for veterinary purposes, the polypeptides of the invention are preferably directed against CX3CR1 from the species to be treated.
  • the immunoglobulin single variable domains comprised in the polypeptides according to the invention are preferably humanized immunoglobulin single variable domains.
  • an immunoglobulin single variable domain can be a domain antibody, i.e. VL or VH antibody, and/or VHH domains as described above, and/or any other sort of immunoglobulin single variable domain, for example camelized VH, provided that these immunoglobulin single variable domains are anti-CX3CR1 immunoglobulin single variable domains.
  • the immunoglobulin single variable domain essentially consists of either a domain antibody sequence or a VHH domain sequence as described above.
  • the immunoglobulin single variable domain essentially consists of a VHH domain sequences.
  • a polypeptide of the present invention comprises two or more anti-CX3CR1 immunoglobulin single variable domains.
  • a polypeptide of the present invention comprises two anti-CX3CR1 immunoglobulin single variable domains, for example anti-CX3CR1 VHHs.
  • the two anti-CX3CR1 immunoglobulin single variable domains in a polypeptide of the present invention have the same amino acid sequence.
  • the two anti-CX3CR1 immunoglobulin single variable domains in a polypeptide of the present invention have different amino acid sequences.
  • the at least two immunoglobulin single variable domains present in a polypeptide of the invention can be linked to each other directly (i.e. without use of a linker) or via a linker.
  • the linker is preferably a linker peptide and will, according to the invention, be selected so as to allow binding of the at least two immunoglobulin single variable domains to CX3CR1, either within one and the same CX3CR1 molecule, or within two different molecules.
  • Suitable linkers will inter alia depend on the epitopes and, specifically, the distance between the epitopes on CX3CR1 to which the immunoglobulin single variable domains bind, and will be clear to the skilled person based on the disclosure herein, optionally after some limited degree of routine experimentation.
  • the two or more anti-CX3CR1 immunoglobulin single variable domains are domain antibodies or VHH domains, they may also be linked to each other via a third domain antibody or VHH domain (in which the two or more immunoglobulin single variable domains may be linked directly to the third domain antibody or VHH domain or via suitable linkers).
  • a third domain antibody or VHH domain may for example be a domain antibody or VHH domain that provides for an increased half-life, as further described herein.
  • the latter domain antibody or VHH domain may be a domain antibody or VHH domain that is capable of binding to a (human) serum protein such as (human) serum albumin or (human) transferrin, as further described herein.
  • the two or more anti-CX3CR1 immunoglobulin single variable domains may be linked in series (either directly or via a suitable linker) and the third (single) domain antibody or VHH domain (which may provide for increased half-life, as decribed above) may be connected directly or via a linker to one of these two or more aforementioned immunoglobulin sequences.
  • Suitable linkers are described herein in connection with specific polypeptides of the invention and may - for example and without limitation - comprise an amino acid sequence, which amino acid sequence preferably has a length of 5 or more amino acids, 7 or more amino acids, 9 or more amino acids, 11 or more amino acids, 15 or more amino acids or at least 17 amino acids, such as about 20 to 40 amino acids.
  • the upper limit is not critical but is chosen for reasons of convenience regarding e.g. biopharmaceutical production of such polypeptides.
  • the linker sequence may be a naturally occurring sequence or a non-naturally occurring sequence. If used for therapeutical purposes, the linker is preferably non-immunogenic in the subject to which the polypeptide of the invention is administered.
  • linker sequences are linkers derived from the hinge region of heavy chain antibodies as described in WO 96/34103 and WO 94/04678 .
  • poly-alanine linker sequences such as Ala-Ala-Ala.
  • linker sequences are Gly/Ser linkers of different length such as (gly x ser y ) z linkers, including (gly 4 ser) 3 , (gly 4 ser) 4 , (gly 4 ser), (gly 3 ser), gly 3 , and (gly 3 ser 2 ) 3 .
  • the linker sequence preferably includes an amino acid residue, such as a cysteine or a lysine, allowing such modification, e.g. PEGylation, in the linker region.
  • a polymer for example a polyethylene glycol (PEG) moiety
  • the linker sequence preferably includes an amino acid residue, such as a cysteine or a lysine, allowing such modification, e.g. PEGylation, in the linker region.
  • linkers are:
  • linker may also be a poly(ethylene glycol) moiety, as shown in e.g. WO04/081026 .
  • Non-limiting examples of polypeptides comprising or consisting of two or more anti-CX3CR1 immunoglobulin single variable domains are given in Table 8a.
  • Table 8a Bivalent anti-CX3CR1 polypeptides CX3CR1 BII007 SEQ ID NO: 267 CX3CR1 BII009 SEQ ID NO: 268 CX3CR1 BII012 SEQ ID NO: 269 CX3CR1 BII016 SEQ ID NO: 270 CX3CR1 BII017 SEQ ID NO: 271 CX3CR1 BII018 SEQ ID NO: 272 CX3CR1 BII019 SEQ ID NO: 273 CX3CR1 BII020 SEQ ID NO: 274 CX3CR1 BII026 SEQ ID NO: 275 CX3CR1 BII027 SEQ ID NO: 276 CX3CR1 BII006 SEQ ID NO: 282
  • the at least two immunoglobulin single variable domains of the polypeptide of the invention are linked to each other via another moiety (optionally via one or two linkers), such as another polypeptide which, in a preferred but non-limiting embodiment, may be a further immunoglobulin single variable domain as already described above.
  • another moiety may either be essentially inactive or may have a biological effect such as improving the desired properties of the polypeptide or may confer one or more additional desired properties to the polypeptide.
  • the moiety may improve the half-life of the protein or polypeptide, and/or may reduce its immunogenicity or improve any other desired property.
  • a polypeptide of the invention includes, especially when used as a therapeutic agent, a moiety which extends the half-life of the polypeptide of the invention in serum or other body fluids of a patient.
  • half-life means the time taken for the serum concentration of the (modified) polypeptide to reduce by 50%, in vivo, for example due to degradation of the polypeptide and/or clearance and/or sequestration by natural mechanisms.
  • the two immunoglobulin single variable domains may be fused to a serum albumin molecule, such as described e.g. in WO01/79271 and WO03/59934 .
  • such half-life extending moiety can be covalently linked or fused to said polypeptide and may be, without limitation, an Fc portion, an albumin moiety, a fragment of an albumin moiety, an albumin binding moiety, such as an anti-albumin immunoglobulin single variable domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin single variable domain, a polyoxyalkylene molecule, such as a polyethylene glycol molecule, an albumin binding peptide, or hydroxyethyl starch (HES) derivatives.
  • Fc portion an albumin moiety, a fragment of an albumin moiety, an albumin binding moiety, such as an anti-albumin immunoglobulin single variable domain, a transferrin binding moiety, such as an anti-transferrin immunoglobulin single variable domain, a polyoxyalkylene molecule, such as a polyethylene glycol molecule, an albumin binding peptide, or hydroxyethyl starch (HES)
  • the polypeptide of the invention comprises a moiety which binds to an antigen found in blood, such as serum albumin, serum immunoglobulins, thyroxine-binding protein, fibrinogen or transferrin, thereby conferring an increased half-life in vivo to the resulting polypeptide of the invention.
  • an antigen found in blood such as serum albumin, serum immunoglobulins, thyroxine-binding protein, fibrinogen or transferrin
  • such moiety is an albumin-binding immunoglobulin and, in particular, an albumin-binding immunoglobulin single variable domain such as an albumin-binding VHH domain.
  • the polypeptide of the invention comprises a moiety which binds to serum albumin, wherein such moiety is an albumin binding peptide, as described e.g. in international patent publications WO2008/068280 and WO2009/127691 .
  • albumin-binding immunoglobulin single variable domain (also called anti-albumin immunoglobulin single variable domain) will preferably bind to human serum albumin and will preferably be a humanized albumin-binding VHH domain.
  • a specifically useful albumin binding VHH domain consists of or contains the amino acid sequence as set forth in any one of SEQ ID NO: 230-232: Table 8b ALB-1 SEQ ID NO: 230 ALB-11 (humanized ALB-1) SEQ ID NO: 231 ALB-2 SEQ ID NO: 232
  • a polypeptide of the invention may be linked to one or more antibody parts, fragments or domains that confer one or more effector functions to the polypeptide of the invention and/or may confer the ability to bind to one or more Fc receptors.
  • the antibody parts may be or may comprise CH2 and/or CH3 domains of an antibody, such as from a heavy chain antibody (as described hereabove) and more preferably from a conventional human 4-chain antibody; specifically, the polypeptide of the invention may be linked to an Fc region, for example from human IgG, from human IgE or from another human Ig.
  • WO 94/04678 describes heavy chain antibodies comprising a Camelid VHH domain or a humanized derivative thereof, in which the Camelidae CH2 and/or CH3 domain have been replaced by human CH2 and/or CH3 domains, so as to provide an immunoglobulin that consists of 2 heavy chains each comprising a - optionally humanized - VHH domain and human CH2 and CH3 domains (but no CH1 domain), which immunoglobulin has the effector function provided by the CH2 and CH3 domains, can function without the presence of any light chains, and has an increased half-life as compared to the corresponding VHH domains without such modification.
  • a polypeptide of the present invention comprises two anti-CX3CR1 VHHs and a VHH capable of binding to serum albumin.
  • the VHHs are fused using linker peptides. Representative examples of such polypeptides of the present invention are shown hereinbelow.
  • a polypeptide of the present invention comprises a first anti-CX3CR1 VHH fused to a first linker peptide, which is itself fused to a VHH capable of binding to serum albumin, which is itself fused to a second linker peptide, which is itself fused to a second anti-CX3CR1 VHH.
  • the first or the second linker peptide is a 9GS linker, in one aspect, the first and the second linker peptide is a 9GS linker.
  • the VHH capable of binding to serum albumin is capable of binding to human serum albumin.
  • the VHH capable of binding to serum albumin has the amino acid sequence set forth in SEQ ID NO: 231.
  • the first and the second anti-CX3CR1 VHH have the same amino acid sequence.
  • the first or the second anti-CX3CR1 VHH has the CDR1, CDR2 and CDR3 set forth in:
  • the first and the second anti-CX3CR1 VHH have the CDR1, CDR2 and CDR3 set forth in:
  • the first or the second anti-CX3CR1 VHH has the amino acid sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224. In one aspect, the first and the second anti-CX3CR1 VHH have the same amino acid sequence, wherein said amino acid sequence is the sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224.
  • Non-limiting examples of polypeptides of the present invention are the polypeptides of any one of SEQ ID NO: 225 to 227, 249 or 277 to 281.
  • Table 9 CX3CR1BII 00312 SEQ ID NO: 225 CX3CR1BII 00313 SEQ ID NO: 226 CX3CR1BII 00314 SEQ ID NO: 227 CX3CR1BII 032 SEQ ID NO: 277 CX3CR1BII 034 SEQ ID NO: 278 CX3CR1BII 036 SEQ ID NO: 249 CX3CR1BII 040 SEQ ID NO: 279 CX3CR1BII 041 SEQ ID NO: 280 CX3CR1BII 042 SEQ ID NO: 281
  • a polypeptide of the present invention comprises an anti-CX3CR1 VHH and a Fc domain.
  • a polypeptide of the present invention comprises an anti-CX3CR1 VHH fused to a linker peptide, which is itself fused to a Fc domain.
  • the linker peptide is a 15GS linker.
  • the Fc domain has the amino acid sequence set forth in SEQ ID NO: 250 or 252.
  • the VHH has the CDR1, CDR2 and CDR3 set forth in:
  • the VHH has the amino acid sequence set forth in any one of SEQ ID NO: 138 to 140 or SEQ ID NO: 222 to 224.
  • the polypeptide is in the form of a dimer, for example wherein the dimer is formed by one or more disulfide bridge.
  • Non-limiting examples of polypeptides of the present invention are the polypeptides of SEQ ID NO: 251, 253 or 254.
  • Table 10 Mouse Fc domain SEQ ID NO: 250 66B02-mFc SEQ ID NO: 251 Human Fc Domain SEQ ID NO: 252 306D-hFc SEQ ID 253 NO: 307D-hFc SEQ ID NO: 254
  • a polypeptide of the invention may be modified to improve its properties.
  • a polypeptide of the present invention may be modified to increase its stability upon storage.
  • a polypeptide of the present invention may be modified to facilitate its expression in a particular host system.
  • the first codon of a polypeptide of the present invention may be modified.
  • a polypeptide of the present invention begins with a glutamic acid (glu) as its first amino acid.
  • a polypeptide of the present invention begins with an aspartic acid (asp) as its first amino acid, for example to reduce pyroglutamate formation at the N-terminus during storage and hence increase product stability.
  • a polypeptide of the present invention begins with an alanine (ala) or a valine (val) as its first amino acid, for example to facilitate the expression of the polypeptide in a prokaryotic expression system, such as Escherichia coli.
  • a prokaryotic expression system such as Escherichia coli.
  • polypeptide of the present invention is characterized by one or more of the following properties:
  • a polypeptide of the present invention has an affinity to human CX3CR1 at an IC50 less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equal to 1nM, as determined by competition FACS.
  • a polypeptide of the present invention has an affinity to human CX3CR1 at an EC50 of less than or equal to 10nM, or less than or equal to 5nM, or less than or equal to 2.5nM or less than or equal to 1nM, as determined by cell binding FACS.
  • a polypeptide of the present invention blocks the binding of human CX3CR1 to human fractalkine at or above 50%, or at or above 60%, or at or above 70%, or at or above 80%, or at or above 90%, or at or above 95% as determined by competition FACS with human fractalkine.
  • a polypeptide of the present invention blocks the binding of human fractalkine to human CX3CR1 at an IC50 of less than or equal to 300nM, or less than or equal to 100nM, or less than or equal to 20nM, or less than or equal to 10nM, less than or equal to 5nM, less than or equal to 2.5nM or less than or equal to 1nM as determined by competition FACS with human fractalkine.
  • a polypeptide of the present invention inhibits fractalkine induced chemotaxis mediated by human CX3CR1 at or above 10%, or at or above 30%, or at or above 40%, or at or above 50%, or at or above 60%, or at or above 70%, or at or above 80%, or at or above 90%.
  • a polypeptide of the present invention inhibits fractalkine induced chemotaxis mediated by human CX3CR1 at an IC50 of less than or equal to 500 nM, or of less than or equal to 100 nM, or less than or equal to 75 nM, or less than or equal to 50 nM, or less than or equal to 10 nM or less than or equal to 5nM.
  • a polypeptide of the present invention inhibits fractalkine induced human CX3CR1 receptor internalization at an IC50 of less than or equal to 10 nM, or less than or equal to 5nM or or less than or equal to 1nM.
  • a half-life extending modification of a polypeptide of the invention comprises attachment of a suitable pharmacologically acceptable polymer, such as straight or branched chain poly(ethylene glycol) (PEG) or derivatives thereof (such as methoxypoly(ethylene glycol) or mPEG).
  • PEG poly(ethylene glycol)
  • derivatives thereof such as methoxypoly(ethylene glycol) or mPEG.
  • any suitable form of PEGylation can be used, such as the PEGylation used in the art for antibodies and antibody fragments (including but not limited to domain antibodies and scFv's); reference is made, for example, to: Chapman, Nat. Biotechnol., 54, 531-545 (2002 ); Veronese and Harris, Adv. Drug Deliv. Rev. 54, 453-456 (2003 ); Harris and Chess, Nat. Rev. Drug. Discov. 2 (2003 ); WO 04/060965 ; and US6,875,841 .
  • reagents for PEGylation of polypeptides are also commercially available, for example from Nektar Therapeutics, USA, or NOF Corporation, Japan, such as the Sunbright ® EA Series, SH Series, MA Series, CA Series, and ME Series, such as Sunbright ® ME-100MA, Sunbright ® ME-200MA, and Sunbright ® ME-400MA.
  • site-directed PEGylation is used, in particular via a cysteine-residue (see for example Yang et al., Protein Engineering 16, 761-770 (2003 )).
  • PEG may be attached to a cysteine residue that naturally occurs in a polypeptide of the invention
  • a polypeptide of the invention may be modified so as to suitably introduce one or more cysteine residues for attachment of PEG, or an amino acid sequence comprising one or more cysteine residues for attachment of PEG may be fused to the N- and/or C-terminus and/or PEG may be attached to a linker region that bridges two or more functional domains of a polypeptide of the invention, all using techniques of protein engineering known per se to the skilled person.
  • a PEG is used with a molecular weight of more than 5 kDa, such as more than 10 kDa and less than 200 kDa, such as less than 100 kDa; for example in the range of 20 kDa to 80 kDa.
  • the invention also encompasses any polypeptide of the invention that has been PEGylated at one or more amino acid positions, preferably in such a way that said PEGylation either (1) increases the half-life in vivo; (2) reduces immunogenicity; (3) provides one or more further beneficial properties known per se for PEGylation; (4) does not essentially affect the affinity of the polypeptide for CX3CR1 (e.g. does not reduce said affinity by more than 50 %, and more preferably not by more than 10%, as determined by a suitable assay, such as those described in the Examples below); and/or (4) does not affect any of the other desired properties of the polypeptides of the invention.
  • Suitable PEG-groups and methods for attaching them, either specifically or non-specifically, will be clear to the skilled person.
  • a PEGylated polypeptide of the invention includes one PEG moiety of linear PEG having a molecular weight of 40 kDa or 60 kDa, wherein the PEG moiety is attached to the polypeptide in a linker region and, specifially, at a Cys residue, for example at position 5 of a GS8-linker peptide as shown in SEQ ID NO:235.
  • PEGylated polypeptides of the invention are PEGylated preferably with one of the PEG reagents as mentioned above, such as "Sunbright ® ME-400MA” as shown in the following chemical formula: which has an average molecular weight of 40 kDa.
  • the present invention provides a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for use as a medicament.
  • the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, or cancer.
  • pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, or cancer.
  • the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis.
  • the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis by preventing and/or reducing the formation of new atherosclerotic lesions or plaques and/or by preventing or slowing progression of existing lesions and plaques.
  • the present invention provides the use of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide for the treatment or prophylaxis of atherosclerosis by changing the composition of the plaques to reduce the risk of plaque rupture and atherothrombotic events.
  • the present invention also provides a method of treating, or reducing the risk of, cardio- and cerebrovascular atherosclerotic disorders, peripheral artery disease, restenosis, diabetic nephropathy, glomerulonephritis, human crescentic glomerulonephritis, IgA nephropathy, membranous nephropathy, lupus nephritis, vasculitis including Henoch-Schonlein purpura and Wegener's granulomatosis, rheumatoid arthritis, osteoarthritis, allograft rejection, systemic sclerosis, neurodegenerative disorders and demyelinating disease, multiple sclerosis (MS), Alzheimer's disease, pulmonary diseases such as COPD, asthma, neuropathic pain, inflammatory pain, or cancer, in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of a polypeptide according to the present invention or a pharmaceutical composition
  • the present invention also provides a method of treating, or reducing the risk of atherosclerosis in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide.
  • the present invention also provides a method of treating, or reducing the risk of atherosclerosis by preventing and/or reducing the formation of new atherosclerotic lesions or plaques and/or by preventing or slowing progression of existing lesions and plaques in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide.
  • the present invention also provides a method of treating, or reducing the risk of atherosclerosis by changing the composition of the plaques so as to reduce the risk of plaque rupture and atherothrombotic events in a person suffering from or at risk of, said disease or condition, wherein the method comprises administering to the person a therapeutically effective amount of a polypeptide of the present invention or a pharmaceutical composition comprising said polypeptide.
  • a polypeptide of the present invention is indicated for use in the treatment or prophylaxis of a disease or disorder that is associated with CX3CR1.
  • a polypeptide of the present invention is indicated for use in the treatment or prophylaxis of diseases or conditions in which modulation of activity at the CX3CR1 receptor is desirable.
  • the present invention also provides a method of treating, or reducing the risk of, diseases or conditions in which antagonism of the CX3CR1 receptor is beneficial which comprises administering to a person suffering from or at risk of, said disease or condition, a polypeptide of the present invention.
  • Prophylaxis is expected to be particularly relevant to the treatment of persons who have suffered a previous episode of, or are otherwise considered to be at increased risk of, the disease or condition in question.
  • Persons at risk of developing a particular disease or condition generally include those having a family history of the disease or condition, or those who have been identified by genetic testing or screening to be particularly susceptible to developing the disease or condition.
  • prevention, treatment and/or alleviation not only comprises preventing and/or treating and/or alleviating the disease, but also generally comprises preventing the onset of the disease, slowing or reversing the progress of disease, preventing or slowing the onset of one or more symptoms associated with the disease, reducing and/or alleviating one or more symptoms associated with the disease, reducing the severity and/or the duration of the disease and/or of any symptoms associated therewith and/or preventing a further increase in the severity of the disease and/or of any symptoms associated therewith, preventing, reducing or reversing any physiological damage caused by the disease, and generally any pharmacological action that is beneficial to the patient being treated.
  • the subject to be treated will be a mammal, and more in particular a human being.
  • the subject to be treated will in particular be a person suffering from, or at risk from, the diseases, disorders or conditions mentioned herein.
  • the above methods of treatment of a disease include the preparation of a medicament for the treatment of said disease.
  • the polypeptides of the invention can be used as an active ingredient in a medicament or pharmaceutical composition intended for the treatment of the above diseases.
  • the invention also relates to the use of a polypeptide of the invention in the preparation of a pharmaceutical composition for the prevention, treatment and/or alleviation of any of the diseases, disorders or conditions mentioned hereinabove.
  • the invention further relates to a polypeptide of the invention for therapeutic or prophylactic use and, specifically, for the prevention, treatment and/or alleviation of any of the diseases, disorders or conditions mentioned hereinabove.
  • the invention further relates to a pharmaceutical composition for the prevention, treatment and/or alleviation of the diseases, disorders or conditions mentioned hereinabove, wherein such composition comprises at least one polypeptide of the invention.
  • polypeptides of the invention and/or the compositions comprising the same can be administered to a patient in need thereof in any suitable manner, depending on the specific pharmaceutical formulation or composition to be used.
  • the polypeptides of the invention and/or the compositions comprising the same can for example be administered intravenously, subcutaneously, intramuscularly, intraperitoneally, transdermally, orally, sublingually (e.g. in the form of a sublingual tablet, spray or drop placed under the tongue and adsorbed through the mucus membranes into the capillary network under the tongue), (intra-)nasally (e.g.
  • a nasal spray and/or as an aerosol topically, by means of a suppository, by inhalation, intravitreally (esp. for the treatment of dry AMD or glaucoma), or any other suitable manner in an effective amount or dose.
  • the polypeptides of the invention and/or the compositions comprising the same are administered according to a regimen of treatment that is suitable for preventing, treating and/or alleviating the disease, disorder or condition to be prevented, treated or alleviated.
  • the clinician will generally be able to determine a suitable treatment regimen, depending on factors such as the disease, disorder or condition to be prevented, treated or alleviated, the severity of the disease, the severity of the symptoms thereof, the specific polypeptide of the invention to be used, the specific route of administration and pharmaceutical formulation or composition to be used, the age, gender, weight, diet, general condition of the patient, and similar factors well known to the clinician.
  • the treatment regimen will comprise the administration of one or more polypeptides of the invention, or of one or more compositions comprising the same, in therapeutically and/or prohylactically effective amounts or doses.
  • the polypeptides of the invention will generally be administered in an amount between 0.005 and 20.0 mg per kilogram of body weight and dose, preferably between 0.05 and 10.0 mg/kg/dose, and more preferably between 0.5 and 10 mg/kg/dose, either continuously (e.g. by infusion) or as single doses (such as e.g. daily, weekly, or monthly doses; cf. below), but can significantly vary, especially, depending on the before-mentioned parameters.
  • compositions containing the polypeptides of the invention may also be administered in similar or slightly lower dosages.
  • the dosage can also be adjusted by the individual physician in the event of any complication.
  • an administration regimen could include long-term, weekly treatment.
  • long-term is meant at least two weeks and preferably months, or years of duration.
  • the efficacy of the polypeptides of the invention, and of compositions comprising the same, can be tested using any suitable in vitro assay, cell-based assay, in vivo assay and/or animal model known per se, or any combination thereof, depending on the specific disease involved.
  • suitable assays and animal models will be clear to the skilled person, and for example include the assays and animal models used in the Examples below.
  • the polypeptides of the invention may be formulated as a pharmaceutical preparation comprising (i) at least one polypeptide of the invention and (ii) at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant, and/or stabilizer, and (iii) optionally one or more further pharmaceutically active polypeptides and/or compounds.
  • pharmaceutically acceptable is meant that the respective material does not show any biological or otherwise undesirable effects when administered to an individual and does not interact in a deleterious manner with any of the other components of the pharmaceutical composition (such as e.g. the pharmaceutically active ingredient) in which it is contained. Specific examples can be found in standard handbooks, such as e.g.
  • the polypeptides of the invention may be formulated and administered in any manner known per se for conventional antibodies and antibody fragments and other pharmaceutically active proteins.
  • the invention relates to a pharmaceutical composition or preparation that contains at least one polypeptide of the invention and at least one pharmaceutically acceptable carrier, diluent, excipient, adjuvant and/or stabilizer, and optionally one or more further pharmaceutically active substances.
  • such a formulation may be in a form suitable for oral administration, for parenteral administration (such as by intravenous, intramuscular, subcutaneous, intrathecal, intracavernosal or intraperitoneal injection or intravenous infusion), for topical administration, for sublingual administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, for transdermal, nasal, intravitreal, rectal or vaginal administration, and the like.
  • parenteral administration such as by intravenous, intramuscular, subcutaneous, intrathecal, intracavernosal or intraperitoneal injection or intravenous infusion
  • topical administration for sublingual administration, for administration by inhalation, by a skin patch, by an implant, by a suppository, for transdermal, nasal, intravitreal, rectal or vaginal administration, and the like.
  • suitable administration forms - which may be solid, semi-solid or liquid, depending on the manner of administration - as well as methods and carriers for use in the preparation thereof
  • compositions for parenteral administration such as intravenous, intramuscular, subcutaneous injection or intravenous infusion may for example be sterile solutions, suspensions, dispersions, emulsions, or powders which comprise the active ingredient and which are suitable, optionally after a further dissolution or dilution step, for infusion or injection.
  • Suitable carriers or diluents for such preparations for example include, without limitation, sterile water and pharmaceutically acceptable aqueous buffers and solutions such as physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution; water oils; glycerol; ethanol; glycols such as propylene glycol, as well as mineral oils, animal oils and vegetable oils, for example peanut oil, soybean oil, as well as suitable mixtures thereof.
  • Solutions of the active compound or its salts may also contain a preservative to prevent the growth of microorganisms, such as antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal (thiomersal), and the like.
  • antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal (thiomersal), and the like.
  • isotonic agents for example, sugars, buffers or sodium chloride.
  • the proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants.
  • Other agents delaying absorption for example, aluminum monostearate and gelatin, may also be added.
  • Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization.
  • the preferred methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions.
  • suitable formulations for therapeutic proteins such as the polypeptides of the invention are buffered protein solutions, such as solutions including the protein in a suitable concentration (such as from 0.001 to 400 mg/ml, preferably from 0.005 to 200 mg/ml, more preferably 0.01 to 200 mg/ml, more preferably 1.0 - 100 mg/ml, such as 1.0 mg/ml (i.v. administration) or 100 mg/ml (s.c. administration) and an aqueous buffer such as:
  • Preferred buffered protein solutions are solutions including about 0.05 mg/ml of the polypeptide of the invention dissolved in 25 mM phosphate buffer, pH 6.5, adjusted to isotonicity by adding 220 mM trehalose.
  • other agents such as a detergent, e.g. 0.02 % Tween-20 or Tween-80, may be included in such solutions.
  • Formulations for subcutaneous application may include significantly higher concentrations of the polypeptide of the invention, such as up to 100 mg/ml or even above 100 mg/ml.
  • the ingredients and the amounts thereof as given above do only represent one, preferred option. Alternatives and variations thereof will be immediately apparent to the skilled person, or can easily be conceived starting from the above disclosure.
  • polypeptides of the invention may also be administered using suitable depot, slow-release or sustained-release formulations, e.g. suitable for injection, using controlled-release devices for implantation under the skin, and/or using a dosing pump or other devices known per se for the administration of pharmaceutically active substances or principles.
  • suitable depot, slow-release or sustained-release formulations e.g. suitable for injection, using controlled-release devices for implantation under the skin, and/or using a dosing pump or other devices known per se for the administration of pharmaceutically active substances or principles.
  • the polypeptides of the invention may be formulated in the form of a gel, cream, spray, drop, patch or film which, if placed on the skin, passes through the skin.
  • polypeptides of the invention can also be easily administered via routes other than parenteral administration and can be easily formulated for such administration.
  • routes other than parenteral administration for example, as described in the international application WO2004/041867 , such polypeptides may be formulated for oral, intranasal, intrapulmonary and transdermal administration.
  • a pharmaceutical combination comprising at least one polypeptide of the invention as disclosed herein and at least one other therapeutic agent selected from the group consisting of statins, antiplatelets, anticoagulants, antidiabetics and antihypertensives.
  • Such pharmaceutical combination may optionally additionally comprise a diluent, excipient, adjuvant and/or stabilizer.
  • two or more substances or principles When two or more substances or principles are to be used as part of a combined treatment regimen, they can be administered via the same route of administration or via different routes of administration, at essentially the same time or at different times (e.g. essentially simultaneously, consecutively, or according to an alternating regime).
  • the substances or principles When the substances or principles are to be administered simultaneously via the same route of administration, they may be administered as different pharmaceutical formulations or compositions or part of a combined pharmaceutical formulation or composition.
  • each of the substances or principles may be administered in the same amount and according to the same regimen as used when the compound or principle is used on its own, and such combined use may or may not lead to a synergistic effect.
  • a further embodiment of the invention is a method for treating the diseases and disorders as set out above, comprising administering to an individual, simultaneously, separately or sequentially, an effective amount of at least one polypeptide of the invention and at least one agent selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.
  • the polypeptide of the invention is prepared to be administered in combination with other drugs used for the treatment of the diseases and disorders set out above, such other drugs being selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive.
  • drugs used for the treatment of the diseases and disorders set out above such drugs being selected from the group consisting of a statin, an antiplatelet, an anticoagulant, an antidiabetic and an antihypertensive are prepared to be administered in combination with the polypeptide of the invention.
  • the polypeptide of the invention is used in combination with a device useful for the administration of the polypeptide, such as a syringe, injector pen, or other device.
  • a device useful for the administration of the polypeptide such as a syringe, injector pen, or other device.
  • a method of diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction comprising the steps of:
  • a method of diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction comprising the steps of:
  • the above diagnostic methods can also be used for monitoring the effectiveness of a therapeutic treatment of a subject.
  • kits for diagnosing a disease, disorder or condition mediated by CX3CR1 dysfunction for use in a method as defined above, such kit comprising at least one polypeptide of the invention and, optionally, one or more media, detection means and/or in vitro or in vivo imaging agents, and, further optionally, instructions of use.
  • Suitable in vivo imaging agents include 99mTc, 111Indium, 123Iodine, and, for magnetic resonance imaging, paramagnetic compounds.
  • the invention further provides a kit comprising at least one polypeptide of the invention and, additionally, one or more other components selected from the group consisting of other drugs used for the treatment of the diseases and disorders as described above, and devices as described above.
  • the invention further provides methods of manufacturing a polypeptide of the invention, such methods generally comprising the steps of:
  • a nucleic acid of the invention can be genomic DNA, cDNA or synthetic DNA (such as DNA with a codon usage that has been specifically adapted for expression in the intended host cell or host organism). According to one embodiment of the invention, the nucleic acid of the invention is in essentially isolated form, as defined hereabove.
  • the nucleic acid of the invention may also be in the form of, be present in and/or be part of a vector, such as for example a plasmid, cosmid or YAC, which again may be in essentially isolated form.
  • the vector may especially be an expression vector, i.e. a vector that can provide for expression of the polypeptide in vitro and/or in vivo (e.g. in a suitable host cell, host organism and/or expression system).
  • Such expression vector generally comprises at least one nucleic acid of the invention that is operably linked to one or more suitable regulatory element(s), such as promoter(s), enhancer(s), terminator(s), and the like.
  • regulatory elements and other elements such as integration factor(s), selection marker(s), signal or leader sequence(s), reporter gene(s), and the like, useful or necessary for expressing polypeptides of the invention, are disclosed e.g. on pp. 131 to 133 of WO2006/040153 .
  • nucleic acids of the invention can be prepared or obtained in a manner known per se (e.g. by automated DNA synthesis and/or recombinant DNA technology), based on the information on the amino acid sequences for the polypeptides of the invention given herein, and/or can be isolated from a suitable natural source.
  • the invention relates to a host or host cell that expresses or is capable of expressing a polypeptide of the invention; and/or that contains a nucleic acid encoding a polypeptide of the invention.
  • said host cells are bacterial cells, yeast cells, fungal cells or mammalian cells.
  • Suitable bacterial cells include cells from gram-negative bacterial strains such as strains of Escherichia coli, Proteus, and Pseudomonas, and gram-positive bacterial strains such as strains of Bacillus, Streptomyces, Staphylococcus, and Lactococcus.
  • Suitable fungal cell include cells from species of Trichoderma, Neurospora, and Aspergillus.
  • Suitable yeast cells include cells from species of Saccharomyces (for example Saccharomyces cerevisiae), Schizosaccharomyces (for example Schizosaccharomyces pombe), Pichia (for example Pichia pastoris and Pichia methanolica), and Hansenula.
  • Suitable mammalian cells include for example CHO cells, BHK cells, HeLa cells, COS cells, NS0 cells, HEK cells, and the like.
  • amphibian cells, insect cells, plant cells, and any other cells used in the art for the expression of heterologous proteins can be used as well.
  • preferred heterologous hosts for the (industrial) production of immunoglobulin single variable domain polypeptides and protein therapeutics containing them include strains of E. coli, Pichia pastoris, and S. cerevisiae that are suitable for large scale expression, production and fermentation, and in particular for large scale (bio-)pharmaceutical expression, production and fermentation.
  • glycosylation pattern obtained i.e. the kind, number and position of residues attached
  • Polypeptides of the invention produced in a cell as set out above can be produced either intracellullarly (e.g. in the cytosol, in the periplasma or in inclusion bodies) and then isolated from the host cells and optionally further purified; or they can be produced extracellularly (secreted into the medium in which the host cells are cultured) and then isolated from the culture medium and optionally further purified.
  • polypeptides such as suitable expression vectors, transformation or transfection methods, selection markers, methods of induction of protein expression, culture conditions, and the like, are known in the art.
  • suitable expression vectors, transformation or transfection methods, selection markers, methods of induction of protein expression, culture conditions, and the like are known in the art.
  • protein isolation and purification techniques useful in a method of manufacture of a polypeptide of the invention are well known to the skilled person.
  • polypeptides of the invention through fermentation in convenient recombinant host organisms such as E. coli and yeast is cost-effective, as compared to conventional antibodies which also require expensive mammalian cell culture facilities. Furthermore, achievable levels of expression are high and yields of the polypeptides of the invention are in the range of 1 to 10 g/l (E. coli) and up to 10 g/l (yeast) and more.
  • CHO and Baf/3 cells overexpressing human or cynomolgus CX3CR1 were generated using techniques known in the art.
  • Cells expressing human CCR2 or CCR5 were also generated using techniques known in the art.
  • the cDNA was cloned into pCDNA3.1(+)-neo for human CX3CR1 whereas pcDNA-DEST40-neo was used for mouse CX3CR1.
  • amino acid sequences of humanCX3CR1 and cynomolgus CX3CR1 are depicted in SEQ ID NO: 255 and 256, respectively.
  • Caki Camel Kidney
  • parental Caki cells were electroporated with pCDNA3.1(+)-neo-hCX3CR1 or pcDNA-DEST40-neo-mCX3CR1, respectively.
  • transfectants were selected by adding 1 mg/mL geneticin (Invitrogen, Carlsbad, CA, USA).
  • Human Embyonic Kidney (HEK293) cells overexpressing human CX3CR1 or cynomolgus CX3CR1 were generated by lipid-mediated transfection with Fugene (Roche) of pCDNA3.1(+)-neo-hCX3CR1 or cyCX3CR1 plasmids, respectively, in the HEK293 parental cell line. These cells were used as transient transfectants and as such not put under selection. In brief, 2*10E6 cells were seeded per T75 and incubated overnight before transfection. After removal of the culture medium, cells were transfected with the respective plasmids (9 ⁇ g) and Fugene (27 ⁇ l) according to manufacturer's instructions. 48 hours post transfection, cells were harvested and frozen for further usage.
  • Example 1 Immunization with CX3CR1 induces a humoral immune response in Ilama
  • 9 llamas (designated No. 368, 369, 370, 381, 382, 384, 312, 313 and 314) were immunized.
  • llamas Six llamas (312, 313, 314, 381, 382 and 384) were immunized with 4 intramuscular injections (2mg/dose at weekly or biweekly intervals) of pVAX1-huCX3CR1 plasmid vector (Invitrogen, Carlsbad, CA, USA). Three llamas (381, 382 and 384) subsequently received 4 subcutaneous injections of human CX3CR1 overexpressing Caki cells which were established as described above. Cells were re-suspended in D-PBS and kept on ice prior to injection.
  • llamas Three additional llamas (designated No. 368, 369 and 370) were immunized according to standard protocols with 4 subcutaneous injections of human CX3CR1 overexpressing Caki cells which were established as described above. Cells were re-suspended in D-PBS and kept on ice prior to injection. Subsequently, these llamas were administered two injections with recombinant CX3CR1 NT/EC3 fragment coupled to BSA (Table 13). Peptides were ordered at NeoMPS (Polypeptidegroup, France) and coupled to BSA according to standard protocols. Table 13 Sequence of peptide fragments used for immunization boost Fragment sequence SEQ ID NO: CX3CR1-NT 228 CX3CR1-EC3 229
  • the first injection was formulated in Complete Freund's Adjuvant (Difco, Detroit, MI, USA), while the subsequent injection was formulated in Incomplete Freund's Adjuvant (Difco, Detroit, MI, USA).
  • immunoglobulins After addition of serum dilutions, specifically bound immunoglobulins were detected using a horseradish peroxidase (HRP)-conjugated goat anti-llama immunoglobulin (Bethyl Laboratories Inc., Montgomery, TX, USA) and a subsequent enzymatic reaction in the presence of the substrate TMB One (3,3',5,5'-tetramentylbenzidine) (Promega, Mannheim, Germany), showing that a significant antibody-dependent immune response against CX3CR1 was induced after the peptide immunizations.
  • HRP horseradish peroxidase
  • Bethyl Laboratories Inc. Montgomery, TX, USA
  • TMB One 3,3',5,5'-tetramentylbenzidine
  • serum titers of cell immunized animals were confirmed by FACS analysis on actively growing human CX3CR1 overexpressing CHO cells.
  • the CX3CR1 serum titer responses for llamas 368, 369 and 370 were determined with serum sampled after 4 cell immunizations (day 49), 4 cell immunizations and 1 peptide boost (day 77) and 4 cell immunizations and 2 peptide boosts (day 81). Cells were harvested and washed before incubation with the serum dilutions.
  • immune tissues as the source of B-cells that produce the heavy-chain antibodies were collected from the immunized llamas.
  • llama 312,313 and 314 two 150-ml blood samples, collected 4 and 8 days after the last antigen injection were collected per animal.
  • llamas 368, 369 and 370 four 150 ml blood samples were collected, 5 and 7 days after the last cell immunization and additionally 4 and 8 days after the last peptide immunization.
  • two lymph node biopsies were taken, 12 days after the last cell immunization and 12 days after the last peptide immunization.
  • llamas 381, 382 and 384 five 150 ml blood samples were collected, 8 days after the last DNA immunization and additionally 4 days after the first cell boost, 8 and 11 days after the second cell boost and 8 days after the last cell immunization. Next to those, one lymph node biopsy was taken, 8 days after the second cell immunization.
  • peripheral blood lymphocytes were prepared using Ficoll-Hypaque according to the manufacturer's instructions (Amersham Biosciences, Piscataway, NJ, USA). From the PBLs and the lymph node biopsy (LN), total RNA was extracted, which was used as starting material for RT-PCR to amplify the VHH encoding DNA segments.
  • libraries were constructed by pooling the total RNA isolated from samples originating from a certain subset of the immunization schedule i.e. after one type of immunization antigen, and for some llamas samples from the different animals were pooled into one library (Table 16).
  • the PCR-amplified VHH repertoire was cloned via specific restriction sites into a vector designed to facilitate phage display of the VHH library.
  • the vector was derived from pUC119 and contains the LacZ promoter, a M13 phage gill protein coding sequence, a resistance gene for ampicillin or carbenicillin, a multiple cloning site and a hybrid gIII-pelB leader sequence (pAX050).
  • the vector encodes a C-terminal c-myc tag and a His6 tag. Phage were prepared according to standard protocols and stored after filter sterilization at 4°C or at -80°C in 20 % glycerol for further use.
  • VHH repertoires obtained from all llamas and cloned as phage library were used in different selection strategies, applying a multiplicity of selection conditions.
  • Variables include i) the presentation form of the CX3CR1 protein (on different cell backgrounds or on liposomes/VLPs), ii) the antigen presentation method (In solution when using cells or coated onto plates when using VLPs), iii) the antigen concentration iv) the orthologue used (human or cynomolgus) v) the number of selection rounds and vi) different elution methods (non-specific via trypsin or specific via the ligand Fractalkine). All solid coated phase selections were done in Maxisorp 96-well plates (Nunc, Wiesbaden, Germany).
  • Phage outputs were used to infect E. coli which were then in turn used to prepare phage for the next selection round (phage rescue) After the second round selection the phage outputs were used to infect E. coli which were then plated on agar plates (LB+carb+glucose 2% ) for analysis of individual VHH clones.
  • agar plates LB+carb+glucose 2%
  • LacZ-controlled VHH expression was induced by adding IPTG (1mM final) in the absence of glucose.
  • Periplasmic extracts (in a volume of ⁇ 80 uL) were prepared according to standard protocols.
  • Periplasmic extracts were screened in a human CX3CR1/human Fractalkine FACS competition assay to assess the blocking capacity of the expressed VHHs.
  • Human CX3CR1 was presented on CHO cells overexpressing CX3CR1. Both a setup using cells harvested from an actively growing culture and a setup using frozen cells was used.
  • As a detection reagent labeled fractalkine was used (R&D Systems, Minneapolis, MN, USA) labeled with alexa647 (A647-Fractalkine) at a degree of labeling of 1.
  • To setup the assay first a titration series of the labeled fractalkine was performed on the CHO-huCX3CR1 cells in order to determine the EC50 value for binding.
  • fractalkine 3 nM
  • EC30 concentration 1 nM was chosen for subsequent screening.
  • 50 ⁇ l of periplasmic extract was added to 6 nM labeled fractalkine (50 ⁇ l) and 200 000 CHO-huCX3CR1 cells. After one hour incubation at 4 C, cells were washed three times before read out was performed on a FACS Array (Becton Dickinson). First a gate was set on the intact cells as determined from the scatter profile. Next, dead cells were gated out by their fluorescence profile from the PI stain (Sigma, St Louis, US).
  • the fluorescence profile from the alexa647 label was determined for each sample and used for calculation of blocking capacity. As controls, conditions were taken along where there was no VHH present in the peri extract or a known irrelevant VHH and samples were included where excess cold fractalkine was included. For each sample the percentage block was determined using the control samples to determine the assay window.
  • VHHs were selected and sequence analysis revealed 120 unique VHHs belonging to 3 different B-cell lineages.
  • the total number of variants found for each B-cell lineage is depicted in Table 17.
  • Table 17 Selection parameters used for the identification of the humanCX3CR1 specific VHH B-cell lineages B-cell lineage
  • Representative VHH ID # variants libraries 9 CX3CR1BII11H11 4 368-PBL1+2+LN-V-100209 13
  • CX3CR1BII18E06 68 368-PBL1+2+LN-V-100209 368-PBL3+4-V-280909 101
  • CX3CR1BII66B02 48 312+313+314-PBL1+2-V-220210 314-PBL1+2-V-180310
  • Inhibitory anti-CX3CR1 VHHs selected from the screening described in Example 4 were further purified and characterized. Selected VHHs were expressed in E. coli TG1 as c-myc, His6-tagged proteins. Expression was induced by addition of 1 mM IPTG and allowed to continue for 4 hours at 37°C. After spinning the cell cultures, periplasmic extracts were prepared by freeze-thawing the pellets. These extracts were used as starting material and VHHs were purified via IMAC and size exclusion chromatography (SEC) resulting in 95% purity as assessed via SDS-PAGE.
  • SEC size exclusion chromatography
  • the blocking capacity towards the ligand fractalkine of the VHHs was evaluated in a human CX3CR1 competition FACS as outlined in Example 4. Either CHO-huCX3CR1 cells, BA/F3-huCX3CR1 cells or transiently transfected HEK293T cells were used. The amount of labeled ligand used in the different competition setups was also varied. The IC 50 values for VHHs blocking the interaction of human fractalkine to human CX3CR1 are depicted in Table 19.
  • a chemotaxis assay was setup using the ChemoTx disposable chamber with 5 ⁇ m poresize (Neuroprobe, Gaithersburg, US). Cells were harvested from an actively growing culture and washed before use in assay medium, RPMI (Gibco, Carlsbad, US) supplemented with 0.1% BSA. The bottom chamber was filled with 320 pM human Fractalkine in a total volume of 300 ⁇ l. Upon application of the membrane, 0.13E6 cells were deposited on top of the membrane in a total volume of 70 ⁇ l. Chemotaxis was allowed for 3 hours at 37°C in a humidified chamber with CO2.
  • a FACS based binding setup was used to evaluate the cynomolgus cross reactivity.
  • the VHHs were incubated with the respective cells for 30 minutes at 4°C followed by three wash steps and subsequently incubated with the detection reagents.
  • detection a mouse anti-cmyc antibody (Serotec, MCA2200) followed by a goat anti-mouse antibody coupled to PE (Jackson 115-116-071) was used, each incubation for 30 minutes at 4°C, followed by three wash steps. Results of the assay are shown in Table 21.
  • a human Fractalkine competition FACS was set up using human or cynomolgus CX3CR1 expressed in HEK293T cells. Both the human and the cynomolgus receptor was transiently transfected in HEK293T cells and transfections were matched by the binding of the labeled ligand, human fractalkine.
  • the competition was evaluated using the EC30 concentration of fractalkine and as such obtained IC50 values are a good estimate of the Ki value, a measure for affinity (Table 22). The experiment was performed as described in Example 4.
  • huCX3CR1 receptor Specificity for the huCX3CR1 receptor was evaluated by performing a FACS binding experiment on CHO-K1 parental cells or CHO cells expressing huCCR2, huCCR5 or msCX3CR1.
  • the VHHs were incubated with the respective cell lines for 30 minutes at 4°C followed by three wash steps and subsequently incubated with the detection reagents.
  • a mouse anti-cmyc antibody Serotec, MCA2200
  • a goat anti-mouse antibody coupled to PE Jackson 115-116-071
  • was used each incubation for 30 minutes at 4°C, followed by three wash steps.
  • a quality control with receptor-specific antibody was included.
  • the highest concentration of each VHH was also incubated with CHO cells expressing huCX3CR1 as a positive control. No binding to msCX3CR1, huCCR2 or huCCR5 could be observed.
  • bivalent molecules were constructed by genetic engineering. Two VHHs were genetically linked together with a 35GS linker in between the two building blocks and subsequently expressed in E.coli as described above for the monovalent VHHs. Different bivalent constructs were made as listed in Table 24.
  • the inhibition of ligand binding to human CX3CR1 was investigated for the different formats as described in Example 4.
  • the BA/F3-huCX3CR1 cell line was used showing stable expression of the human CX3CR1 receptor.
  • the alexa647 labeled ligand fractalkine was used at its EC30 concentration and thereby obtained IC50 values are reflective of the Ki values.
  • An overview of the obtained data is shown in Table 25.
  • linker length used in a bivalent format can impact drastically on the obtained potency, different linker lengths were evaluated.
  • Coding sequences for the formatted VHH were cloned into an in-house constructed plasmid allowing expression in Pichia pastoris and secretion into the cultivation medium.
  • the expression vector was derived from pPICZa (Invitrogen) and contained the AOX1 promotor for tightly regulated, methanol induced expression, a restance gene for Zeocin TM , a multicloning site and the ⁇ -factor secretion signal.
  • pPICZa Invitrogen
  • AOX1 promotor for tightly regulated, methanol induced expression, a restance gene for Zeocin TM , a multicloning site and the ⁇ -factor secretion signal.
  • the potency of these different formats was evaluated using the ligand competition assay as described above. Seeing that the ligand concentration used is below the EC50 value, the obtained IC50 values are equivalent to the Ki values.
  • the obtained Ki for the different formats is summarized in Table 30.
  • HSA human serum albumin
  • Construct ID Cell line IC50 (M) % block Repeats CX3CR1BII032 BA/F3-huCX3CR1 1.4 E-9 100 2 CX3CR1BII034 BA/F3-huCX3CR1 1.3 E-9 100 2 CX3CR1BII036 BA/F3-huCX3CR1 1.4 E-9 100 2
  • HSA human immunosensing protein
  • concentration HSA used was again a 50 fold excess over the highest concentration of construct used and constructs were loaded with HSA for 30 minutes before start of the assay.
  • the assay buffer was also supplemented with HSA such that HSA is present during the entire span of the experiment.
  • the disposable ChemoTx chamber with 5 ⁇ m poresize was used. Cells were harvested from an actively growing culture and washed before use in assay medium, RPMI (Gibco, Carlsbad, US) supplemented with 0.1% BSA and 62.5 ⁇ M HSA (Sigma, A8763).
  • the bottom chamber was filled with 320 pM human Fractalkine in a total volume of 300 ⁇ l.
  • 0.13E6 cells were deposited on top of the membrane in a total volume of 70 ⁇ l.
  • Chemotaxis was allowed for 3 hours at 37 C in a humidified chamber with CO2. After this incubation period, the membrane was removed and cells in the bottom chamber were resuspended.
  • the amount of ATP present in well was determined using the CellTiter-Glo kit (Promega, Madison WI, USA). Read out was performed on an Envision (Perkin Elmer, Waltham, MA, USA) with the standard factory settings for luminescence read out. Titration series were performed in triplicate and each plate contained control samples in triplicate as well. As control, a sample without VHH was included as well as a sample where no human Fractalkine was added to the bottom chamber. The obtained IC50 values are listed in Table 31.
  • polypeptides were evaluated for their ability to inhibit the internalization of A647-Fractalkine in CHO huCX3CR1 cells. Briefly, 1E4 cells/well were plated in black clear bottom, 96 well plates (BD, Franklin Lakes, NJ, USA) and grown overnight. The cells were washed once and then equilibrated in assay buffer (HBSS with calcium and magnesium (Gibco) supplemented with 10 mM HEPES and 0.1% BSA). The formatted polypeptide constructs were added and the plates were incubated for 15 minutes at 37 C.
  • assay buffer HBSS with calcium and magnesium (Gibco) supplemented with 10 mM HEPES and 0.1% BSA.
  • A647-Fractalkine was then added at a final concentration of 8 nM and the cells were incubated for 60 minutes at 37 C. The media was removed and the cells were fixed for 10 minutes with 3.7% formaldehyde solution (Polysciences, Warrington, PA, USA). The cells were rinsed once with PBS and the nuclei were labeled with Hoechst dye (Life Technologies, Grand Island, NY, USA). To quantitate the internalized labeled Fractalkine, the cells were imaged using the BD Pathway bioimaging system. Image segmentation was performed by identifying the labeled cell nucleus and drawing a 3 pixel ring around that mask. Mean A647 intensity was measured in the cytoplasmic ring.
  • An anti-CX3CR1 formatted bivalent half-life extended polypeptide is devoid of agonist activity
  • CX3CR1BII036 was evaluated for induction of calcium influx in the CHO huCX3CR1 cells. Fractalkine mediated increases in cytosolic calcium levels in these cells in a CX3CR1 dependent manner and CX3CR1BII036 inhibited this response.
  • the CHO huCX3CR1 cells were plated at 5E4 cells/well in black clear bottom, 96 well plates (BD) and grown overnight. The cells were incubated with Calcium-4 dye/2 mM probenicid (Molecular Devices, Sunnyvale, CA, USA) in HBSS supplemented with 20 mM HEPES for 60 minutes at 37 °C.
  • CX3CR1BII036 was preincubated with the cells for 15 minutes prior to the addition of Fractalkine at its EC80 value. Calcium mobilization was monitored on a FLIPR Tetra system (Molecular Devices) as per the manufacturer's instructions.
  • CX3CR1BII036 was used in place of Fractalkine stimulation. While CX3CR1BII036 inhibited Fractalkine mediated calcium influx with an IC50 of 1.3 nM, no increase in cytosolic calcium levels were observed when the polypeptide alone was added at concentrations up to 1 ⁇ M.
  • the 66B02 VHH domain was produced as a fusion protein with a mouse IgG2 Fc domain (66B02-mFc).
  • An aspartic acid to alanine mutation (D265A) was incorporated in the CH2 domain to abrogate potential Fc-mediated effector function in this construct (Baudino, J. Immunol., 181, 6664-6669 (2008)).
  • 66B02-mFc was expressed in HEK293T cells or NS0 cells and purified by Protein A affinity chromatography followed by ion exchange chromatography. This molecule was tested for activity utilizing the assay formats described in Example 7.
  • Table 33 66B02-mFc Activity Assay Cell line IC50 (M) Repeats Ligand competition BA/F3-huCX3CR1 5.7 E-10 2 Chemotaxis BA/F3-huCX3CR1 8.9 E-10 3 Ligand internalization CHO-huCX3CR1 5.2 E-10 5 Calcium influx CHO-huCX3CR1 9.8 E-10 5
  • Example 9 Inhibition of plaque progression in a mouse atherosclerosis model bivalent half-life extended polypeptides
  • a human CX3CR1 knock-in mouse line (hu CX3CR1 KI) was generated at TaconicArtemis (Koeln, Germany) to enable testing of these molecules in mouse disease models.
  • TaconicArtemis Koeln, Germany
  • a strategy was employed that allowed the expression of the human chemokine receptor under the control of the corresponding mouse promoter while disrupting the expression of the endogenous mouse protein.
  • a targeting vector was constructed where the mouse CX3CR1 coding region in exon 2 was replaced with the complete human CX3CR1 open reading frame and flanked by selection markers and loxP sites.
  • the targeting vector was introduced into mouse ES cells and clones that had successfully undergone homologous recombination were used to generate chimeric mice. These mice were bred to highly efficient Flp-deleter mice to achieve removal of the selection marker and germline transmission. The resulting hu CX3CR1 KI mice in a C57BL/6 background were then crossed to Apo E -/- mice (The Jackson Laboratory, Bar Harbor, Maine, USA) to generate hu CX3CR1 KI Apo E -/- mice.
  • the Apo E -/- mouse model provides a robust method to elicit extensive atherosclerotic plaque formation that is grossly similar to the human disease with respect to the site-specific localization of plaque formation, histological composition, and the known risk factors (cholesterol, inflammation, hypertension, etc).
  • mice Female hu CX3CR1KI ApoE -/- mice were fed a high fat/high cholesterol diet containing 1.5% cholesterol for 16 weeks beginning at four weeks of age. After 10 weeks, the animals were administered by i.p. injection vehicle (20 mM NaCitrate pH 6.0, 115 mM NaCl), 10 mg/kg 66B02-mFc once or twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6 weeks. The animals were anesthetized by gas anaesthesia and perfused with 0.9% saline. The descending aorta to the ileac bifurcation was carefully removed and fixed in formalin.
  • Table 34 Quantification of Plaque Size in the Descending Aorta in female hu CX3CR1 KI Apo E -/- Mice Group Dose # Animals % Plaque Area % Reduction in Plaque Area Control (10 weeks) N/A 6 3.4 N/A Control (16 weeks) Vehicle 17 14.8 N/A 66B02-mFc 10 mg/kg (1xweek) 17 13.0 16 66B02-mFc 10 mg/kg (2x/week) 17 10.3 39 (p ⁇ 0.05) CX3CR1BII036 30 mg/kg (2x/week) 17 10.1 41 (p ⁇ 0.05)
  • 66B02-mFc and CX3CR1BII036 significantly inhibited plaque progression when dosed twice weekly. This correlated with coverage as plasma levels of these molecules could be confirmed to be maintained throughout the study. For once weekly dosing of 66B02-mFc, detectable plasma levels were not maintained and this correlated with the lack of significant efficacy observed after 6 weeks of treatment. Neither molecule significantly affected plasma cholesterol or triglyceride levels.
  • VHH sequence optimization parental wild type VHH sequences are mutated to yield VHH sequences that are more identical to human VH3-JH germline consensus sequences.
  • Specific amino acids in the framework regions that differ between the VHH and the human VH3-JH germline consensus are altered to the human counterpart in such a way that the protein structure, activity and stability are kept intact.
  • a glycosylation site was predicted at position 52. Therefore two libraries were constructed; one for position 52 and one for position 53, which was designed to include all possible amino acids at the respective position.
  • the libraries were screened as periplasmic extracts in a ligand competition FACS. First, a dilution series was made of periplasmic material from the parental sequence and three dilutions were selected for further screening. A first dilution point (two fold) was chosen to give full block of the ligand interaction whereas the other two dilution points (128 and 512 fold) should result in 70 % and 40% block respectively.
  • a sequence optimized anti-CX3CR1 half-life extended polypeptide is devoid of agonist activity
  • CX3CR1BII00313 was evaluated for induction of calcium influx in the CHO huCX3CR1 cells. While preincubation with CX3CR1B1100313 inhibited Fractalkine-mediated calcium influx with an IC50 of 1.3 nM, no increase in cytosolic calcium levels were observed when the polypeptide alone was added at concentrations up to 1 ⁇ M.
  • the CX3CR1BII00306 and CX3CR1BII00307 sequence optimized VHH domains were produced as fusion proteins with a human IgG1 Fc domain (306D-hFc and 307D-hFc). Two mutations were incorporated in the CH2 domain to abrogate potential Fc-mediated effector function in this construct. 306D-hFc and 307D-hFc were expressed in HEK293T cells or NS0 cells and purified by Protein A affinity chromatography followed by ion exchange chromatography. These molecules were tested for functional activity utilizing the assay formats described in Example 7.
  • Table 42 Activity of hFc Fusion Proteins Assay Cell line 306D-hFc IC50 306D-hFc Repeats 307D-hFc IC50 307D-hFc Repeats Ligand competition BA/F3-huCX3CR1 6.9 E-10 2 7.0 E-10 2 Chemotaxis BA/F3-huCX3CR1 2.9 E-9 2 3.0 E-9 3 Ligand internalization CHO-huCX3CR1 4.8 E-10 3 3.7 E-10 3 Calcium influx CHO-huCX3CR1 1.3 E-9 3 3.2 E-9 3
  • Example 13 Inhibition of plaque progression in a mouse atherosclerosis model by a sequence optimized anti-CX3CR1 Nanobody
  • mice Female hu CX3CR1KI ApoE -/- mice were fed a high fat/high cholesterol diet containing 1.5% cholesterol for 16 weeks beginning at four weeks of age. After 10 weeks, the animals were administered by i.p. injection vehicle (20 mM NaCitrate pH 6.0, 115 mM NaCl), 30 mg/kg CX3CR1BII00313 once or twice per week or 30 mg/kg CX3CR1BII036 twice per week for 6 weeks. The animals were sacrificed and the percentage of plaque area in the descending aorta was quantitated as described above.
  • Table 43 Quantification of Plaque Size in the Descending Aorta in female hu CX3CR1 KI Apo E -/- Mice Group Dose # Animals % Plaque Area % Reduction in Plaque Area Control (10 weeks) N/A 6 2.1 N/A Control (16 weeks) Vehicle 18 12.0 N/A CX3CR1BII00313 30 mg/kg (1xweek) 17 10.7 13 CX3CR1BII00313 30 mg/kg (2x/week) 18 5.9 62 (p ⁇ 0.01) CX3CR1BII036 30 mg/kg (2x/week) 17 6.8 52 (p ⁇ 0.01)
  • CX3CR1BII00313 and CX3CR1BII036 significantly inhibited plaque progression when dosed twice weekly. This correlated with coverage as plasma levels of these molecules could be confirmed to be maintained throughout the study. For once weekly dosing of CX3CR1BII00313, detectable plasma levels were not maintained and this correlated with the lack of significant efficacy observed after 6 weeks of treatment. Neither molecule significantly affected plasma cholesterol or triglyceride levels.
  • Example 14 Nanobody binding to primary human and cynomolgus monkey CD14+ cells in whole blood
  • CX3CR1BII00313 was demonstrated to compete for the binding of A647 labeled CX3CR1BII018 (A647-018) to CD14+ cells in a competition FACS assay in whole blood.
  • a mouse anti-human CD14 antibody conjugated with eFluor 450 (eBioscience, San Diego, CA, USA) was diluted 1:10 in EDTA treated whole blood from a healthy human donor.
  • CX3CR1BII00313 was demonstrated to compete for the binding of A647 labeled CX3CR1BII018 (A647-018) to CD14+ cells in a competition FACS assay in cynomolgus monkey whole blood.
  • the method used was analogous to that outlined above except the final concentration of A647-018 was 3 nM (the EC80 of A647-018 binding) and ACK lysing buffer (Life Technologies) was used instead of the 1-Step Fix/Lyse solution. The cells were resuspended in Stain buffer supplemented with 1% formaldehyde prior to analysis.
  • CX3CR1BII00313 was administered as a 2 mg/ml solution in citrate buffer (20 mM sodium citrate/115 mM sodium chloride, pH 6.0). Blood samples were collected over 6 weeks from a peripheral vein into serum separator tubes for PK analysis.
  • Serum samples were analyzed using a MSD (Meso Scale Discovery) format. Briefly, a biotinylated anti-Nanobody antibody was bound to a MSD standard streptavidin plate (Meso Scale Discovery, Rockville, MD, USA). The plates were washed with 0.05% Tween 20 in phosphate buffered saline and blocked with 5% w/v of SeraCare BSA (SeraCare Life Sciences, Milford, MA, USA) prior to incubation with serum samples. CX3CR1BII00313 was detected utilizing a sulfolabeled anti-Nanobody Nanobody and the plates were analyzed on a Sector Imager 2400 (Meso Scale Discovery).
  • Varying concentrations of CX3CR1BII0313 from 5000 to 0.5 ng/ml in 5% monkey serum were used as standards. Target engagement was assessed by monitoring levels of free CX3CR1 on CD14+ gated monocytes. This assay was analogous to the competition FACS assay summarized in Example 14 except no additional CX3CR1BII00313 was added. Serum samples were also monitored for the presence of primate anti-human antibodies (PAHA) as they may impact assessment of PK and free CX3CR1.
  • PAHA primate anti-human antibodies
  • CX3CR1BII0313 was captured over streptavidin sensors. Pooled naive monkey serum was then used as a negative control to calculate cut-off value (defined as two fold above the average binding signal of naive sera). All serum samples were diluted 20 fold in buffer and the PAHA response was determined to be positive if the binding signal was greater than the cut-off value.
  • the present disclosure further contains the following items:

Landscapes

  • Health & Medical Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Chemical & Material Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Immunology (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • Biophysics (AREA)
  • General Engineering & Computer Science (AREA)
  • Wood Science & Technology (AREA)
  • Neurology (AREA)
  • Neurosurgery (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Pulmonology (AREA)
  • Cardiology (AREA)
  • Rheumatology (AREA)
  • Heart & Thoracic Surgery (AREA)
  • Urology & Nephrology (AREA)
  • Psychiatry (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Vascular Medicine (AREA)
  • Orthopedic Medicine & Surgery (AREA)
EP22168124.0A 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1 Pending EP4050027A3 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
US201261603622P 2012-02-27 2012-02-27
PCT/US2013/027580 WO2013130381A1 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1
EP13709641.8A EP2820046B1 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1

Related Parent Applications (2)

Application Number Title Priority Date Filing Date
EP13709641.8A Division EP2820046B1 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1
EP13709641.8A Division-Into EP2820046B1 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1

Publications (2)

Publication Number Publication Date
EP4050027A2 true EP4050027A2 (fr) 2022-08-31
EP4050027A3 EP4050027A3 (fr) 2022-11-30

Family

ID=47884535

Family Applications (2)

Application Number Title Priority Date Filing Date
EP13709641.8A Active EP2820046B1 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1
EP22168124.0A Pending EP4050027A3 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1

Family Applications Before (1)

Application Number Title Priority Date Filing Date
EP13709641.8A Active EP2820046B1 (fr) 2012-02-27 2013-02-25 Polypeptides se liant au cx3cr1

Country Status (28)

Country Link
US (6) US9035029B2 (fr)
EP (2) EP2820046B1 (fr)
JP (1) JP6101715B2 (fr)
KR (1) KR102008136B1 (fr)
CN (3) CN115925938A (fr)
AP (1) AP2014007791A0 (fr)
AR (1) AR090158A1 (fr)
AU (1) AU2013226340B2 (fr)
CA (1) CA2862182A1 (fr)
CL (1) CL2014002072A1 (fr)
EA (1) EA028183B1 (fr)
EC (1) ECSP14020402A (fr)
GE (1) GEP201706773B (fr)
HK (1) HK1205154A1 (fr)
IL (3) IL291571B2 (fr)
IN (1) IN2014DN05756A (fr)
MD (1) MD4548C1 (fr)
MX (1) MX349192B (fr)
NZ (1) NZ627260A (fr)
PE (1) PE20141859A1 (fr)
PH (1) PH12014501912A1 (fr)
SG (1) SG11201405283SA (fr)
TN (1) TN2014000360A1 (fr)
TW (1) TWI598361B (fr)
UA (1) UA115781C2 (fr)
UY (1) UY34644A (fr)
WO (1) WO2013130381A1 (fr)
ZA (1) ZA201405021B (fr)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9101674B2 (en) 2010-03-29 2015-08-11 Vib Vzw Targeting and in vivo imaging of tumor-associated macrophages
US9556273B2 (en) 2010-03-29 2017-01-31 Vib Vzw Anti-macrophage mannose receptor single variable domains for targeting and in vivo imaging of tumor-associated macrophages
WO2013130381A1 (fr) 2012-02-27 2013-09-06 Boehringer Ingelheim International Gmbh Polypeptides se liant au cx3cr1
WO2015026884A1 (fr) * 2013-08-21 2015-02-26 Boehringer Ingelheim International Gmbh Agents d'imagerie de ciblage de cx3cr1 et leur utilisation dans le diagnostic et le traitement de maladie
RU2609627C2 (ru) * 2014-09-26 2017-02-02 Закрытое Акционерное Общество "Биокад" Высокоаффинные и агрегационно стабильные антитела на основе вариабельных доменов vl и производного vhh
BR112018001161A2 (pt) 2015-07-23 2018-09-18 Inhibrx Lp proteínas de fusão de ligação a gitr multiespecíficas e multivalentes
WO2017123673A2 (fr) 2016-01-11 2017-07-20 Inhibrx Lp Protéines de fusion multivalentes et multispécifiques fixant ox40
US12128102B2 (en) 2016-03-08 2024-10-29 Takeda Pharmaceutical Company Limited Constrained conditionally activated binding proteins
SG11201807548SA (en) * 2016-03-08 2018-09-27 Maverick Therapeutics Inc Inducible binding proteins and methods of use
US20200071408A1 (en) * 2016-11-29 2020-03-05 National Center Of Neurology And Psychiatry Prophylactic agent, onset-suppressing agent or therapeutic agent for progressive immune demyelinating diseases
WO2018134815A2 (fr) * 2017-01-17 2018-07-26 Yeda Research And Development Co. Ltd. Procédés de traitement de maladies neurodégénératives par induction de cellules de microglie associées à une maladie (dam)
WO2019051102A2 (fr) 2017-09-08 2019-03-14 Maverick Therapeutics, Inc. Protéines de liaison à activation conditionnelle restreinte
AU2019321490A1 (en) 2018-08-13 2021-02-18 Inhibrx Biosciences, Inc. Ox40-binding polypeptides and uses thereof
JP2022524338A (ja) 2019-03-05 2022-05-02 武田薬品工業株式会社 拘束され、条件的に活性化された結合タンパク質
CN110675421B (zh) * 2019-08-30 2022-03-15 电子科技大学 基于少量标注框的深度图像协同分割方法
WO2022165434A2 (fr) * 2021-02-01 2022-08-04 The General Hospital Corporation Anticorps améliorés par fc pour la prévention et le traitement d'une infection par le virus ebola
WO2024017915A1 (fr) 2022-07-18 2024-01-25 Ablynx N.V. Composés de liaison à cx3cr1, leurs utilisations et procédés associés
WO2024135794A1 (fr) * 2022-12-22 2024-06-27 株式会社カイオム・バイオサイエンス Anticorps anti-cx3cr1 humain

Citations (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (fr) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulines exemptes de chaines legeres
WO1996034103A1 (fr) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Fragments variables d'immunoglobulines et leur utilisation dans un but therapeutique ou veterinaire
WO1998049185A1 (fr) 1997-04-28 1998-11-05 Fmc Corporation Canaux chlorure actives par gaba chez des lepidopteres
WO2001079271A1 (fr) 2000-04-12 2001-10-25 Principia Pharmaceutical Corporation Proteines fusionnees a l'albumine
WO2003002609A2 (fr) 2001-06-28 2003-01-09 Domantis Limited Ligand
WO2003059934A2 (fr) 2001-12-21 2003-07-24 Human Genome Sciences, Inc. Proteines de fusion d'albumine
WO2004041867A2 (fr) 2002-11-08 2004-05-21 Ablynx N.V. Procede d'administration de polypeptides therapeutiques et polypeptides associes
WO2004060965A2 (fr) 2002-12-31 2004-07-22 Nektar Therapeutics Al, Corporation Polymeres a terminaison maleimide hydrolytiquement stables
WO2004081026A2 (fr) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
US6875841B2 (en) 2001-07-31 2005-04-05 Nof Corporation Polyoxyalkylene derivative and process of producing the same
WO2006040153A2 (fr) 2004-10-13 2006-04-20 Ablynx N.V. Nanocorps™ contre la proteine beta-amyloide et polypeptides les renfermant pour le traitement de maladies degeneratives neurales, telles que la maladie d'alzheimer
WO2006122786A2 (fr) 2005-05-18 2006-11-23 Ablynx Nv Nanocorpstm; utilises contre le facteur-alpha de necrose tumorale
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
WO2008068280A1 (fr) 2006-12-05 2008-06-12 Ablynx N.V. Peptides capables de se lier à des protéines sériques
WO2009109635A2 (fr) 2008-03-05 2009-09-11 Ablynx Nv Nouveaux complexes dimères de fixation à des antigènes, procédés de fabrication et utilisations associés
WO2009127691A1 (fr) 2008-04-17 2009-10-22 Ablynx N.V. Peptides capables de se lier à des protéines sériques et composés, constructions et polypeptides les comprenant

Family Cites Families (16)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2000273378A1 (en) * 2000-02-18 2001-08-27 Millennium Pharmaceuticals, Inc. Therapeutic methods that target fractalkine or cx3cr1
US20020192212A1 (en) 2001-03-19 2002-12-19 Toshio Imai Uses of anti-CX3CR1 antibody, anti-fractalkine antibody and fractalkine
US20050069962A1 (en) 2001-10-12 2005-03-31 Archer Robert M Antibody complexes and methods for immunolabeling
US20060160076A1 (en) 2002-06-10 2006-07-20 Meatabolex, Inc. Methods of treating and diagnosing diabetes with cx3cr1 modulators
WO2005103684A2 (fr) 2004-04-20 2005-11-03 Bayer Healthcare Ag Diagnostics et methodes therapeutiqeus pour des maladies associees au recepteur 1 de la chimiokine cx3c
JP4979388B2 (ja) 2004-10-29 2012-07-18 エーザイ・アール・アンド・ディー・マネジメント株式会社 炎症性疾患治療剤
WO2007019541A2 (fr) 2005-08-08 2007-02-15 Onconon, Llc. Compositions d'anticorps, methodes de traitement de maladie neoplasique et methodes de regulation de la fertilite
WO2007024715A2 (fr) * 2005-08-19 2007-03-01 Abbott Laboratories Immunoglobuline a deux domaines variables et utilisations de celle-ci
CN101646689A (zh) * 2006-09-08 2010-02-10 埃博灵克斯股份有限公司 具有长半衰期的血清清蛋白结合蛋白
CA2726652A1 (fr) 2008-06-05 2009-12-10 Ablynx N.V. Sequences d'acides amines dirigees contre des proteines d'enveloppe d'un virus, et polypeptides comprenant ces sequences destines au traitement de maladies virales
WO2010070394A1 (fr) 2008-12-17 2010-06-24 Universite Pierre Et Marie Curie-Paris Vi Modulateurs du récepteur cx3cr1 et leurs utilisations thérapeutiques
WO2011044377A2 (fr) 2009-10-09 2011-04-14 Cell Point, Llc Conjugués chélate-ligand de ciblage pour l'imagerie cardiovasculaire
EP2486056A1 (fr) * 2009-10-09 2012-08-15 Ablynx N.V. Domaine variable unique immunoglobulinique dirigé contre cxcr4 humain et autres protéines associées à des cellules, et procédés d'obtention
JP6076913B2 (ja) * 2010-12-07 2017-02-08 ドレクセル ユニバーシティ 癌からの転移を阻害する方法
WO2013130381A1 (fr) 2012-02-27 2013-09-06 Boehringer Ingelheim International Gmbh Polypeptides se liant au cx3cr1
WO2015026884A1 (fr) 2013-08-21 2015-02-26 Boehringer Ingelheim International Gmbh Agents d'imagerie de ciblage de cx3cr1 et leur utilisation dans le diagnostic et le traitement de maladie

Patent Citations (17)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1994004678A1 (fr) 1992-08-21 1994-03-03 Casterman Cecile Immunoglobulines exemptes de chaines legeres
WO1996034103A1 (fr) 1995-04-25 1996-10-31 Vrije Universiteit Brussel Fragments variables d'immunoglobulines et leur utilisation dans un but therapeutique ou veterinaire
WO1998049185A1 (fr) 1997-04-28 1998-11-05 Fmc Corporation Canaux chlorure actives par gaba chez des lepidopteres
WO2001079271A1 (fr) 2000-04-12 2001-10-25 Principia Pharmaceutical Corporation Proteines fusionnees a l'albumine
WO2003002609A2 (fr) 2001-06-28 2003-01-09 Domantis Limited Ligand
US6875841B2 (en) 2001-07-31 2005-04-05 Nof Corporation Polyoxyalkylene derivative and process of producing the same
WO2003059934A2 (fr) 2001-12-21 2003-07-24 Human Genome Sciences, Inc. Proteines de fusion d'albumine
WO2004041867A2 (fr) 2002-11-08 2004-05-21 Ablynx N.V. Procede d'administration de polypeptides therapeutiques et polypeptides associes
WO2004060965A2 (fr) 2002-12-31 2004-07-22 Nektar Therapeutics Al, Corporation Polymeres a terminaison maleimide hydrolytiquement stables
WO2004081026A2 (fr) 2003-06-30 2004-09-23 Domantis Limited Polypeptides
WO2006040153A2 (fr) 2004-10-13 2006-04-20 Ablynx N.V. Nanocorps™ contre la proteine beta-amyloide et polypeptides les renfermant pour le traitement de maladies degeneratives neurales, telles que la maladie d'alzheimer
WO2006122786A2 (fr) 2005-05-18 2006-11-23 Ablynx Nv Nanocorpstm; utilises contre le facteur-alpha de necrose tumorale
WO2006122787A1 (fr) 2005-05-18 2006-11-23 Ablynx Nv Proteines de liaison a l'albumine serique
WO2008020079A1 (fr) 2006-08-18 2008-02-21 Ablynx N.V. Séquences d'acides aminés dirigées contre l'il-6r et polypeptides les contenant utilisés pour le traitement de maladies et de troubles associés au signal médié par il-6
WO2008068280A1 (fr) 2006-12-05 2008-06-12 Ablynx N.V. Peptides capables de se lier à des protéines sériques
WO2009109635A2 (fr) 2008-03-05 2009-09-11 Ablynx Nv Nouveaux complexes dimères de fixation à des antigènes, procédés de fabrication et utilisations associés
WO2009127691A1 (fr) 2008-04-17 2009-10-22 Ablynx N.V. Peptides capables de se lier à des protéines sériques et composés, constructions et polypeptides les comprenant

Non-Patent Citations (24)

* Cited by examiner, † Cited by third party
Title
"Antibody Engineering", vol. 2, 2010, SPRINGER VERLAG
BARBAS ET AL., PROC. NAT. ACAD. SCI, USA, vol. 91, 1994, pages 3809 - 3813
BAUDINO, J. IMMUNOL., vol. 181, 2008, pages 6664 - 6669
CHAPMAN, NAT. BIOTECHNOL., vol. 54, 2002, pages 531 - 545
COMBADIERE, CIRCULATION, vol. 107, 2003, pages 1009
GHILARDI, STROKE, vol. 35, 2004, pages 1276
HAMERS-CASTERMAN CATARHOUCH TMUYLDERMANS SROBINSON GHAMERS CSONGA EBBENDAHMAN NHAMERS R: "Naturally occurring antibodies devoid of light chains", NATURE, vol. 363, 1993, pages 446 - 448, XP002535892, DOI: 10.1038/363446a0
HARRISCHESS, NAT. REV. DRUG. DISCOV., vol. 2, 2003
HAWKINS ET AL., J. MOL. BIOL., vol. 226, no. 3, 1992, pages 889 - 896
HOLT, L.J. ET AL.: "Domain antibodies: proteins for therapy", TRENDS IN BIOTECHNOLOGY, vol. 21, no. 11, 2003, pages 484 - 490, XP004467495, DOI: 10.1016/j.tibtech.2003.08.007
JACKSON ET AL., J. IMMUNOL., vol. 154, no. 7, 1995, pages 3310 - 9
KS JOHNSONRE HAWKINS: "Affinity maturation of antibodies using phage display", 1996, OXFORD UNIVERSITY PRESS
LESNIK, J. CLIN. INVEST, vol. 111, 2003, pages 333
LEWIN: "Remington's Pharmaceutical Sciences", 1990, MACK PUBLISHING COMPANY
MARKS ET AL., BIOTECHNOLOGY, vol. 10, 1992, pages 779 - 783
MCDERMOTT, CIRC RES, vol. 89, 2001, pages 401
MCDERMOTT, J. CLIN. INVEST., vol. 111, 2003, pages 1241
RIECHMANNMUYLDERMANS, J. IMMUNOL. METHODS, vol. 231, 1999, pages 25 - 38
SHIER ET AL., GENE, vol. 169, 1995, pages 147 - 155
VERONESEHARRIS, ADV. DRUG DELIV., vol. 54, 2003, pages 453 - 456
WARD, E.S. ET AL.: "Binding activities of a repertoire of single immunoglobulin variable domains secreted from Escherichia coli", NATURE, vol. 341, 1989, pages 544 - 546
WONG, CARDIOVASC. PATH., vol. 11, 2002, pages 332
YANG ET AL., PROTEIN ENGINEERING, vol. 16, 2003, pages 761 - 770
YELTON ET AL., IMMUNOL, vol. 155, 1995, pages 1994 - 2004

Also Published As

Publication number Publication date
WO2013130381A1 (fr) 2013-09-06
KR20140130685A (ko) 2014-11-11
CN104245735B (zh) 2018-02-23
US11384151B2 (en) 2022-07-12
IL311502A (en) 2024-05-01
UY34644A (es) 2013-09-02
SG11201405283SA (en) 2014-10-30
CL2014002072A1 (es) 2015-01-23
US20150239971A1 (en) 2015-08-27
PH12014501912A1 (en) 2014-11-24
JP2015509368A (ja) 2015-03-30
US20160355595A1 (en) 2016-12-08
IL291571A (en) 2022-05-01
ECSP14020402A (es) 2015-11-30
US20230046772A1 (en) 2023-02-16
KR102008136B1 (ko) 2019-08-08
AU2013226340A1 (en) 2014-07-31
US20180030141A1 (en) 2018-02-01
MX349192B (es) 2017-07-18
MD4548C1 (ro) 2018-08-31
US9035029B2 (en) 2015-05-19
EP4050027A3 (fr) 2022-11-30
IL234018B2 (en) 2023-05-01
UA115781C2 (uk) 2017-12-26
IL291571B1 (en) 2024-04-01
EA028183B1 (ru) 2017-10-31
CA2862182A1 (fr) 2013-09-06
TN2014000360A1 (en) 2015-12-21
TW201348256A (zh) 2013-12-01
AP2014007791A0 (en) 2014-07-31
MX2014009864A (es) 2014-10-24
US9783612B2 (en) 2017-10-10
BR112014021080A2 (pt) 2017-07-04
US20130224224A1 (en) 2013-08-29
ZA201405021B (en) 2024-09-25
US9458235B2 (en) 2016-10-04
HK1205154A1 (en) 2015-12-11
CN115925938A (zh) 2023-04-07
IL234018A0 (en) 2014-09-30
CN108610421A (zh) 2018-10-02
CN104245735A (zh) 2014-12-24
GEP201706773B (en) 2017-11-27
EP2820046B1 (fr) 2022-06-15
EP2820046A1 (fr) 2015-01-07
IL234018B1 (en) 2023-01-01
US20240279349A2 (en) 2024-08-22
AU2013226340B2 (en) 2017-06-22
AR090158A1 (es) 2014-10-22
US20240026013A2 (en) 2024-01-25
MD4548B1 (ro) 2018-01-31
IN2014DN05756A (fr) 2015-04-10
US10385134B2 (en) 2019-08-20
JP6101715B2 (ja) 2017-03-22
TWI598361B (zh) 2017-09-11
IL291571B2 (en) 2024-08-01
US20190322753A1 (en) 2019-10-24
NZ627260A (en) 2015-10-30
MD20140104A2 (ro) 2015-02-28
CN108610421B (zh) 2022-08-19
PE20141859A1 (es) 2014-12-17
EA201400964A1 (ru) 2015-02-27

Similar Documents

Publication Publication Date Title
US11384151B2 (en) CX3CR1-binding polypeptides comprising immunoglobulin single variable domains
US8937164B2 (en) Biological materials related to CXCR7
EP2313436B1 (fr) Séquences d acides aminés dirigées contre des récepteurs de désactiveurs multicibles et polypeptides
JP2019503167A (ja) Ｃｄ３８に対する抗原結合性ポリペプチド
WO2004041863A2 (fr) Anticorps a domaine unique diriges contre un interferon gamma et leurs utilisations
EP1558646A2 (fr) Anticorps a domaine unique diriges contre un interferon gamma et leurs utilisations
JP2011516520A (ja) Ｎｏｔｃｈ経路に指向性を有するアミノ酸配列及びその使用
US20060034833A1 (en) Single domain antibodies directed against interferron-gamma and uses therefor
EP3682898A1 (fr) Composition pharmaceutique comprenant un anticorps se liant de manière spécifique à l'extrémité n-terminale de la lysyl-arnt synthétase en tant que principe actif pour la prévention ou le traitement d'une maladie liée à la migration des cellules immunitaires
OA17072A (en) CX3CR1-binding polypeptides.
BR122021000901B1 (pt) Polipeptídeo, seu método de fabricação, célula hospedeira bacteriana, de levedura ou fúngica, composição farmacêutica, kit de diagnóstico, e método de diagnóstico
BR122021000924B1 (pt) Polipeptídeo, seu método de fabricação, célula hospedeira bacteriana, de levedura ou fúngica, composição farmacêutica, kit de diagnóstico, e método de diagnóstico
BR112014021080B1 (pt) Polipeptídeo, seu método de fabricação, célula hospedeira bacteriana, de levedura ou fúngica, composição farmacêutica, kit de diagnóstico, e método de diagnóstico

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: REQUEST FOR EXAMINATION WAS MADE

17P Request for examination filed

Effective date: 20220413

AC Divisional application: reference to earlier application

Ref document number: 2820046

Country of ref document: EP

Kind code of ref document: P

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

PUAL Search report despatched

Free format text: ORIGINAL CODE: 0009013

AK Designated contracting states

Kind code of ref document: A3

Designated state(s): AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LI LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR

RIC1 Information provided on ipc code assigned before grant

Ipc: A61P 9/00 20060101ALI20221027BHEP

Ipc: C07K 16/00 20060101AFI20221027BHEP

REG Reference to a national code

Ref country code: HK

Ref legal event code: DE

Ref document number: 40077734

Country of ref document: HK